Pyrrolobenzodiazepines used to treat proliferative diseases

ABSTRACT

Pyrrolobenzodiazepine dimers I having a C2-C3 double bond and an aryl group at the C2 position on one monomer unit, and a C2-C3 double bond and either a conjugated double or triple bond at the C2 position or an alkyl group at the C2 position on the other monomer unit, and conjugates of these compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a national stage filing under 35 U.S.C. 371of International Application No. PCT/US2011/032668, filed on Apr. 15,2011, which claims priority to U.S. Provisional Patent Application No.61/324,453, filed on Apr. 15, 2010. These applications are incorporatedherein by reference in their entireties.

The present invention relates to pyrrolobenzodiazepines (PBDs), inparticular pyrrolobenzodiazepine dimers having a C2-C3 double bond andan aryl group at the C2 position on one monomer unit, and a C2-C3 doublebond and either a conjugated double or triple bond at the C2 position oran alkyl group at the C2 position on the other monomer unit.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11 position which is the electrophilic centre responsiblefor alkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11(1975); Hurley and Needham-VanDeventer, Acc. Chem. Res., 19, 230-237(1986)). Their ability to form an adduct in the minor groove, enablesthem to interfere with DNA processing, hence their use as antitumouragents.

It has been previously disclosed that the biological activity of thismolecules can be potentiated by joining two PBD units together throughtheir C8/C′-hydroxyl functionalities via a flexible alkylene linker(Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992);Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBDdimers are thought to form sequence-selective DNA lesions such as thepalindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., etal., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al.,Biochemistry, 44, 4135-4147) which is thought to be mainly responsiblefor their biological activity. One example of a PBD dimmer, SG2000(SJG-136):

has recently completed Phase I clinical trials in the oncology area andis about to enter Phase II (Gregson, S., et al., J. Med. Chem., 44,737-748 (2001); Alley, M. C., et al., Cancer Research, 64, 6700-6706(2004); Hartley, J. A., et al., Cancer Research, 64, 6693-6699 (2004)).

More recently, the present inventors have previously disclosed in WO2005/085251, dimeric PBD compounds bearing C2 aryl substituents, such asSG2202 (ZC-207):

and in WO2006/111759, bisulphites of such PBD compounds, for exampleSG2285 (ZC-423):

These compounds have been shown to be highly useful cytotoxic agents(Howard, P. W., et al., Bioorg. Med. Chem. (2009), doi:10.1016/j.bmc1.2009.09.012).

Due to the manner in which these highly potent compounds act incross-linking DNA, these molecules have been made symmetrically. Thisprovides for straightforward synthesis, either by constructing the PBDmoieties simultaneously having already formed the dimer linkage, or byreacting already constructed PBD moieties with the dimer linking group.

Co-pending International Application PCT/GB2009/002498, filed 16 Oct.2009, discloses unsymmetrical dimeric PBD compound bearing aryl groupsin the C2 position of each monomer, where one of these groups bears asubstituent designed to provide an anchor for linking the compound toanother moiety.

DISCLOSURE OF THE INVENTION

The present inventors have developed further unsymmetrical dimeric PBDcompounds bearing an aryl group in the C2 position of one monomer, saidaryl group bearing a substituent designed to provide an anchor forlinking the compound to another moiety, and either a unsaturated bondconjugated to the C2-C3 double bond or an alkyl group in the othermonomer unit.

The present invention comprises a compound with the formula I:

wherein:

-   R² is of formula II:

-   where A is a C₅₋₇ aryl group, X is selected from the group    comprising: OH, SH, CO₂H, COH, N═C═O, NHNH₂, CONHNH₂,

-   NHR^(N), wherein R^(N) is selected from the group comprising H and    C₁₋₄ alkyl, and either:-   (i) Q¹ is a single bond, and Q² is selected from a single bond and    —Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH    and n is from 1 to 3; or-   (ii) Q¹ is —CH═CH—, and Q² is a single bond;-   R¹² is selected from:-   (iia) C₁₋₅ saturated aliphatic alkyl;-   (iiib) C₃₋₆ saturated cycloalkyl;-   (iic)

-    wherein each of R²¹, R²² and R²³ are independently selected from H,    C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl,    where the total number of carbon atoms in the R¹² group is no more    than 5;-   (iid)

-    wherein one of R^(25a) and R^(25b) is H and the other is selected    from: phenyl, which phenyl is optionally substituted by a group    selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and-   (iie)

-    where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl;    C₂₋₃ alkynyl; cyclopropyl; phenyl, which phenyl is optionally    substituted by a group selected from halo, methyl, methoxy; pyridyl;    and thiophenyl;-   R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,    NHR, NRR′, nitro, Me₃Sn and halo;-   where R and R′ are independently selected from optionally    substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups;-   R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro,    Me₃Sn and halo; either:-   (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl;-   (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the    nitrogen and carbon atoms to which they are bound; or-   (c) R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3 and M is a    monovalent pharmaceutically acceptable cation;-   R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one    or more heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄    alkyl), and/or aromatic rings, e.g. benzene or pyridine;-   Y and Y′ are selected from O, S, or NH;-   R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷    and R⁹ respectively and R^(10′) and-   R^(11′) are the same as R¹⁰ and R¹¹, wherein if R¹¹ and R^(11′) are    SO_(z)M, M may represent a divalent pharmaceutically acceptable    cation.

A second aspect of the present invention provides the use of a compoundof the first aspect of the invention in the manufacture of a medicamentfor treating a proliferative disease. The second aspect also provides acompound of the first aspect of the invention for use in the treatmentof a proliferative disease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

A third aspect of the present invention comprises a compound of formulaII:

wherein:

-   R² is of formula II:

-   where A is a C₅₋₇ aryl group, X is selected from the group    comprising: OH, SH, CO₂H, COH, N═C═O, NHNH₂, CONHNH₂,

-    NHR^(N), wherein R^(N) is selected from the group comprising H and    C₁₋₄ alkyl, and either:-   (i) Q¹ is a single bond, and Q² is selected from a single bond and    —Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH    and n is from 1 to 3; or-   (ii) Q¹ is —CH═CH—, and Q² is a single bond;-   R¹² is selected from:-   (iia) C₁₋₅ saturated aliphatic alkyl;-   (iib) C₃₋₆ saturated cycloalkyl;-   (iic)

-   10 wherein each of R²¹, R²² and R²³ are independently selected from    H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl,    where the total number of carbon atoms in the R¹² group is no more    than 5;-   (iid)

-    wherein one of R^(25a) and R^(25b) is H and the other is selected    from: phenyl, which phenyl is optionally substituted by a group    selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and-   (iie)

-    where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl;    C₂₋₃ alkynyl: cyclopropyl; phenyl, which phenyl is optionally    substituted by a group selected from halo, methyl, methoxy; pyridyl;    and thiophenyl;-   R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,    NHR, NRR′, nitro, Me₃Sn and halo;-   where R and R′ are independently selected from optionally    substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₆₋₂₀ aryl groups;-   R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro,    Me₃Sn and halo; either:-   (a) R¹⁰ is carbamate nitrogen protecting group, and R¹¹ is    O-Prot^(O), wherein Prot^(O) is an oxygen protecting group;-   (b) R¹⁰ is a hemi-aminal nitrogen protecting group and R¹¹ is an oxo    group;-   R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one    or more heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄    alkyl), and/or aromatic rings, e.g. benzene or pyridine;-   Y and Y′ are selected from O, S, or NH;-   R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷    and R⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and    R¹¹.

A fourth aspect of the present invention comprises a method of making acompound of formula I from a compound of formula II by deprotection ofthe imine bond.

The unsymmetrical dimeric PBD compounds of the present invention aremade by different strategies to those previously employed in makingsymmetrical dimeric PBD compounds. In particular, the present inventorshave developed a method which involves adding each each C2 substituentto a symmetrical PBD dimer core in separate method steps. Accordingly, afifth aspect of the present invention provides a method of making acompound of the first or third aspect of the invention, comprising atleast one of the method steps set out below.

In a sixth aspect, the present invention relates to Conjugatescomprising dimers of PBDs linked to a targeting agent, wherein a PBD isa dimer of formula I (supra).

In some embodiments, the Conjugates have the following formula III:L-(LU-D)_(p)  (III)wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linkerunit and D is a Drug unit comprising a PBD dimer. The subscript p is aninteger of from 1 to 20. Accordingly, the Conjugates comprise a Ligandunit covalently linked to at least one Drug unit by a Linker unit. TheLigand unit, described more fully below, is a targeting agent that bindsto a target moiety. The Ligand unit can, for example, specifically bindto a cell component (a Cell Binding Agent) or to other target moleculesof interest. Accordingly, the present invention also provides methodsfor the treatment of, for example, various cancers and autoimmunedisease. These methods encompass the use of the Conjugates wherein theLigand unit is a targeting agent that specifically binds to a targetmolecule. The Ligand unit can be, for example, a protein, polypeptide orpeptide, such as an antibody, an antigen-binding fragment of anantibody, or other binding agent, such as an Fc fusion protein.

The PBD dimer D is of formula I, except that X is selected from thegroup comprising: O, S, C(═O), C═, NH(C═O), NHNH, CONHNH,

NR^(N), wherein R^(N) is selected from the group comprising H and C₁₋₄alkyl.

BRIEF DESCRIPTION OF FIGURE

FIG. 1 shows the effect of a conjugate of the invention on a tumour.

DEFINITIONS

Pharmaceutically Acceptable Cations

Examples of pharmaceutically acceptable monovalent and divalent cationsare discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), whichis incorporated herein by reference.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cationsinclude, but are not limited to, alkali metal ions such as Na⁺ and K⁺.Examples of pharmaceutically acceptable divalent inorganic cationsinclude, but are not limited to, alkaline earth cations such as Ca²⁺ andMg²⁺. Examples of pharmaceutically acceptable organic cations include,but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substitutedammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl”includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussedbelow.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:

-   cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane    (C₆), cycloheptane (C₇), methylcyclopropane (C₄),    dimethylcyclopropane (C₅), methylcyclobutane (C₅),    dimethylcyclobutane (C₆), methylcyclopentane (C₆),    dimethylcyclopentane (C₇) and methylcyclohexane (C₇);    -   unsaturated monocyclic hydrocarbon compounds:

-   cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅), cyclohexene    (C₆), methylcyclopropene (C₄), dimethylcyclopropene (C₅),    methylcyclobutene (C₅), dimethylcyclobutene (C₆), methylcyclopentene    (C₆), dimethylcyclopentene (C₇) and methylcyclohexene (C₇); and    -   saturated polycyclic hydrocarbon compounds:        norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

-   N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)    (C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅),    2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine    (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);-   O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅),    oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆),    dihydropyran (C₆), pyran (C₆), oxepin (C₇);-   S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene)    (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);-   O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);-   O₃: trioxane (C₆);-   N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline    (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);-   N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅),    tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆),    tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆);-   N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);-   N₂O₁: oxadiazine (C₆);-   O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,-   N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₆₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein,pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

-   N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);-   O₁: furan (oxole) (C₅);-   S₁: thiophene (thiole) (C₅);-   N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);-   N₂O₁: oxadiazole (furazan) (C₅);-   N₃O₁: oxatriazole (C₅);-   N₁S₁: thiazole (C₅), isothiazole (C₅);-   N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),    pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,    cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);-   N₃: triazole (C₅), triazine (C₆); and,-   N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene        (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁),        phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),        thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),        phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groupsinclude, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇alkylacyl or C₁₋₇ alkanoyl), aC₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)CH₂CH₃, —OC(═O)OC(CH₃)₃, and—OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇alkylamino), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case of a“cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyl,and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and ═NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of sulfinyloxy groups include, but are not limited to,—OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group.

Examples of sulfonyloxy groups include, but are not limited to,—OS(═O)₂CH₃ (mesylate) and —OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂,—OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to abidentate moiety obtained by removing two hydrogen atoms, either bothfrom the same carbon atom, or one from each of two different carbonatoms, of a hydrocarbon compound having from 3 to 12 carbon atoms(unless otherwise specified), which may be aliphatic or alicyclic, andwhich may be saturated, partially unsaturated, or fully unsaturated.Thus, the term “alkylene” includes the sub-classes alkenylene,alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are notlimited to, —(CH₂)_(n)— where n is an integer from 3 to 12, for example,—CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂—(pentylene) and —CH₂CH₂CH₂CH—₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂ alkylene groups include, but arenot limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—,—CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and—CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene, and alkynylene groups) include, but are not limited to,—CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—,—CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—,—CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—C≡C—CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups(C₃₋₁₂alkenylene and alkynylene groups) include, but are not limited to,—C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C—CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentylene (e.g.cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene;3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Oxygen protecting group: the term “oxygen protecting group” refers to amoiety which masks a hydroxy group, and these are well known in the art.A large number of suitable groups are described on pages 23 to 200 ofGreene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis,3^(rd) Edition, John Wiley & Sons, Inc., 1999, which is incorporatedherein by reference. Classes of particular interest include silyl ethers(e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g.acetate).

Carbamate nitrogen protecting group: the term “carbamate nitrogenprotecting group” pertains to a moiety which masks the nitrogen in theimine bond, and these are well known in the art. These groups have thefollowing structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference.

Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogenprotecting group” pertains to a group having the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference.Conjugates

The present invention provides Conjugates comprising a PBD dimerconnected to a Ligand unit via a Linker Unit. In one embodiment, theLinker unit includes a Stretcher unit (A), a Specificity unit (L¹), anda Spacer unit (L²). The Linker unit is connected at one end to theLigand unit and at the other end to the PBD dimer compound.

In one aspect, such a Conjugate is shown below in formula IIIa:L-(A¹ _(a)-L¹ _(s)-L² _(y)-D)_(p)  (IIIa)

-   -   wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)-L² _(y)- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit,    -   a is 1 or 2,    -   L¹- is a Specificity unit,    -   s is an integer ranging from 1 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is an PBD dimer; and    -   p is from 1 to 20.

In another aspect, such a Conjugate is shown below in formula IIIb:

-   -   Also illustrated as:        L-(A¹ _(a)-L² _(y)(-L¹ _(s))-D)_(p)  (Ib)    -   wherein:    -   L is the Ligand unit; and    -   -A¹ _(a)-L¹ _(s)(L² _(y))- is a Linker unit (LU), wherein:    -   -A¹- is a Stretcher unit linked to a Stretcher unit (L²),    -   a is 1 or 2,    -   L¹- is a Specificity unit linked to a Stretcher unit (L²),    -   s is an integer ranging from 0 to 12,    -   -L²- is a Spacer unit,    -   y is 0, 1 or 2;    -   -D is a PBD dimer; and    -   p is from 1 to 20.        Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or

may relate to a single aspect. The preferences may be combined togetherin any combination.

In one embodiment, the Conjugate has the formula:L-(A¹ _(a)-L¹ _(s)-L² _(y)-D)_(p)

-   -   wherein L, A¹, a, L¹, s, L², D and p are as described above.

In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit, which is a covalent bond, a        self-immolative group or together with —OC(═O)— forms a        self-immolative group, and L² optional.

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:CBA-A¹ _(a)-L¹ _(s)-L² _(y)-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit (D), CBA is the Cell Binding Agent, L¹ is a Specificity        unit, A¹ is a Stretcher unit connecting L¹ to the Cell Binding        Agent, L² is a Spacer unit which is a covalent bond or a        self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is        0 or 1 or 2.

In the embodiments illustrated above, L¹ can be a cleavable Specificityunit, and may be referred to as a “trigger” that when cleaved activatesa self-immolative group (or self-immolative groups) L², when aself-immolative group(s) is present. When the Specificity unit L¹ iscleaved, or the linkage (i.e., the covalent bond) between L¹ and L² iscleaved, the self-immolative group releases the Drug unit (D).

In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA)that specifically binds to a target molecule on the surface of a targetcell. An exemplary formula is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        (D), CBA is the Cell Binding Agent, L¹ is a Specificity unit        connected to L², A¹ is a Stretcher unit connecting L² to the        Cell Binding Agent, L² is a self-immolative group, and a is 1 or        2, s is 1 or 2, and y is 1 or 2.

In the various embodiments discussed herein, the nature of L¹ and L² canvary widely. These groups are chosen on the basis of theircharacteristics, which may be dictated in part, by the conditions at thesite to which the conjugate is delivered. Where the Specificity unit L¹is cleavable, the structure and/or sequence of L¹ is selected such thatit is cleaved by the action of enzymes present at the target site (e.g.,the target cell). L¹ units that are cleavable by changes in pH (e.g.acid or base labile), temperature or upon irradiation (e.g. photolabile)may also be used. L¹ units that are cleavable under reducing oroxidising conditions may also find use in the Conjugates.

In some embodiments, L¹ may comprise one amino acid or a contiguoussequence of amino acids. The amino acid sequence may be the targetsubstrate for an enzyme.

In one embodiment, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase. For example, L¹may be cleaved by a lysosomal protease, such as a cathepsin.

In one embodiment, L² is present and together with —C(═O)O— forms aself-immolative group or self-immolative groups. In some embodiments,—C(═O)O— also is a self-immolative group.

In one embodiment, where L¹ is cleavable by the action of an enzyme andL² is present, the enzyme cleaves the bond between L¹ and L², wherebythe self-immolative group(s) release the Drug unit.

L¹ and L², where present, may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH, and    -   —O— (a glycosidic bond).

An amino group of L¹ that connects to L² may be the N-terminus of anamino acid or may be derived from an amino group of an amino acid sidechain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to L² may be the C-terminus of anamino acid or may be derived from a carboxyl group of an amino acid sidechain, for example a glutamic acid amino acid side chain.

A hydroxy group of L¹ that connects to L² may be derived from a hydroxygroup of an amino acid side chain, for example a serine amino acid sidechain.

In one embodiment, —C(═O)O— and L² together form the group:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to the L¹,        Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The        phenylene ring is optionally substituted with one, two or three        substituents as described herein.

In one embodiment, Y is NH.

In one embodiment, n is 0 or 1. Preferably, n is 0.

Where Y is NH and n is 0, the self-immolative group may be referred toas a p-aminobenzylcarbonyl linker (PABC).

The self-immolative group will allow for release of the Drug unit (i.e.,the asymmetric PBD) when a remote site in the linker is activated,proceeding along the lines shown below (for n=0):

-   -   where the asterisk indicates the attachment to the Drug, L* is        the activated form of the remaining portion of the linker and        the released Drug unit is not shown. These groups have the        advantage of separating the site of activation from the Drug.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above. Each phenylene ring is optionally substituted with one,        two or three substituents as described herein. In one        embodiment, the phenylene ring having the Y substituent is        optionally substituted and the phenylene ring not having the Y        substituent is unsubstituted.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or        CR.

In one embodiment, D is N.

In one embodiment, D is CH.

In one embodiment, E is O or S.

In one embodiment, F is CH.

In a preferred embodiment, the covalent bond between L¹ and L² is acathepsin labile (e.g., cleavable) bond.

In one embodiment, L¹ comprises a dipeptide. The amino acids in thedipeptide may be any combination of natural amino acids and non-naturalamino acids. In some embodiments, the dipeptide comprises natural aminoacids. Where the linker is a cathepsin labile linker, the dipeptide isthe site of action for cathepsin-mediated cleavage. The dipeptide thenis a recognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys-, Val-Cit or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those described byDubowchik et al., which is incorporated herein by reference.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below. Protected amino acid sequences are cleavable byenzymes. For example, a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those aminoacids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;    -   Asn: Trt, Xan;    -   Asp: Bzl, t-Bu;    -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;    -   Glu: Bzl, t-Bu;    -   Gln: Trt, Xan;    -   His: Boc, Dnp, Tos, Trt;    -   Lys: Boc, Z—Cl, Fmoc, Z;    -   Ser: Bzl, TBDMS, TBDPS;    -   Thr: Bz;    -   Trp: Boc;    -   Tyr: Bzl, Z, Z—Br.

In one embodiment, —X₂— is connected indirectly to the Drug unit. Insuch an embodiment, the Spacer unit L² is present.

In one embodiment, the dipeptide is used in combination with aself-immolative group(s) (the Spacer unit). The self-immolative group(s)may be connected to —X₂—.

Where a self-immolative group is present, —X₂— is connected directly tothe self-immolative group. In one embodiment, —X₂— is connected to thegroup Y of the self-immolative group. Preferably the group —X₂—CO— isconnected to Y, where Y is NH.

—X₁— is connected directly to A¹. In one embodiment, —X₁— is connecteddirectly to A¹. Preferably the group NH—X₁— (the amino terminus of X₁)is connected to A¹. A¹ may comprise the functionality —CO— thereby toform an amide link with —X₁—.

In one embodiment, L¹ and L² together with —OC(═O)— comprise the group—X₁—X₂-PABC-. The PABC group is connected directly to the Drug unit. Inone example, the self-immolative group and the dipeptide together formthe group -Phe-Lys-PABC-, which is illustrated below:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of L¹ or the point of attachment to A¹.        Preferably, the wavy line indicates the point of attachment to        A¹.

Alternatively, the self-immolative group and the dipeptide together formthe group -Val-Ala-PABC-, which is illustrated below:

-   -   where the asterisk and the wavy line are as defined above.

In another embodiment, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A¹, Y        is a covalent bond or a functional group, and E is a group that        is susceptible to cleavage thereby to activate a self-immolative        group.

E is selected such that the group is susceptible to cleavage, e.g., bylight or by the action of an enzyme. E may be —NO₂ or glucuronic acid(e.g., β-glucuronic acid). The former may be susceptible to the actionof a nitroreductase, the latter to the action of a β-glucuronidase.

The group Y may be a covalent bond.

The group Y may be a functional group selected from:

-   -   —C(═O)—    -   —NH—    -   —O—    -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   SO₂, and    -   —S—.

The group Y is preferably —NH—, —CH₂—, —O—, and —S—.

In some embodiments, L¹ and L² together with —OC(═O)— represent:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, the wavy line indicates the point of attachment to A, Y is        a covalent bond or a functional group and E is glucuronic acid        (e.g., β-glucuronic acid). Y is preferably a functional group        selected from —NH—.

In some embodiments, L¹ and L² together represent:

-   -   where the asterisk indicates the point of attachment to the        remainder of L² or the Drug unit, the wavy line indicates the        point of attachment to A¹, Y is a covalent bond or a functional        group and E is glucuronic acid (e.g., β-glucuronic acid). Y is        preferably a functional group selected from —NH—, —CH₂—, —O—,        and —S—.

In some further embodiments, Y is a functional group as set forth above,the functional group is linked to an amino acid, and the amino acid islinked to the Stretcher unit A¹. In some embodiments, amino acid isβ-alanine. In such an embodiment, the amino acid is equivalentlyconsidered part of the Stretcher unit.

The Specificity unit L¹ and the Ligand unit are indirectly connected viathe Stretcher unit.

L¹ and A¹ may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the group A¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the connection between the Ligand unit and A¹ isthrough a thiol residue of the Ligand unit and a maleimide group of A¹.

In one embodiment, the connection between the Ligand unit and A¹ is:

-   -   where the asterisk indicates the point of attachment to the        remaining portion of A¹, L¹, L² or D, and the wavy line        indicates the point of attachment to the remaining portion of        the Ligand unit. In this embodiment, the S atom is typically        derived from the Ligand unit.

In each of the embodiments above, an alternative functionality may beused in place of the malemide-derived group shown below:

-   -   where the wavy line indicates the point of attachment to the        Ligand unit as before, and the asterisk indicates the bond to        the remaining portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the wavy line indicates point of attachment to the Ligand        unit, and the asterisk indicates the bond to the remaining        portion of the A¹ group, or to L¹, L² or D.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with a Ligand unit (e.g., a Cell BindingAgent), is selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   —S—,    -   —S—S—,    -   —CH₂C(═O)—    -   —C(═O)CH₂—,    -   ═N—NH—, and    -   —NH—N═.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the Ligand unit, is selected from:

-   -   where the wavy line indicates either the point of attachment to        the Ligand unit or the bond to the remaining portion of the A¹        group, and the asterisk indicates the other of the point of        attachment to the Ligand unit or the bond to the remaining        portion of the A¹ group.

Other groups suitable for connecting L¹ to the Cell Binding Agent aredescribed in WO 2005/082023.

In one embodiment, the Stretcher unit A¹ is present, the Specificityunit L¹ is present and Spacer unit L² is absent. Thus, L¹ and the Drugunit are directly connected via a bond. Equivalently in this embodiment,L² is a bond.

L¹ and D may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,

—OC(═O)—,

-   -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

In one embodiment, L¹ and D are preferably connected by a bond selectedfrom:

-   -   —C(═O)NH—, and    -   —NHC(═O)—.

In one embodiment, L¹ comprises a dipeptide and one end of the dipeptideis linked to D. As described above, the amino acids in the dipeptide maybe any combination of natural amino acids and non-natural amino acids.In some embodiments, the dipeptide comprises natural amino acids. Wherethe linker is a cathepsin labile linker, the dipeptide is the site ofaction for cathepsin-mediated cleavage. The dipeptide then is arecognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is-Phe-Lys- or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those describedabove.

In one embodiment, L¹-D is:

-   -   where —NH—X₁—X₂—CO is the dipeptide, —NH— is part of the Drug        unit, the asterisk indicates the point of attachment to the        remainder of the Drug unit, and the wavy line indicates the        point of attachment to the remaining portion of L¹ or the point        of attachment to A¹. Preferably, the wavy line indicates the        point of attachment to A¹.

In one embodiment, the dipeptide is valine-alanine and L¹-D is:

-   -   where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is phenylalnine-lysine and L¹-D is:

-   -   where the asterisk, —NH— and the wavy line are as defined above.

In one embodiment, the dipeptide is valine-citrulline.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3        or 7.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A¹-L¹ is:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, S is        a sulfur group of the Ligand unit, the wavy line indicates the        point of attachment to the rest of the Ligand unit, and n is 0        to 6. In one embodiment, n is 5.

In one embodiment, the group L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, S is        a sulfur group of the Ligand unit, the wavy line indicates the        point of attachment to the remainder of the Ligand unit, and n        is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, S is        a sulfur group of the Ligand unit, the wavy line indicates the        point of attachment to the remainder of the Ligand unit, n is 0        or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m        is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to D, the        wavy line indicates the point of attachment to the Ligand unit,        n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the groups L-A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the Stretcher unit is an acetamide unit, having theformula:

-   -   where the asterisk indicates the point of attachment to the        remainder of the Stretcher unit, L¹ or D, and the wavy line        indicates the point of attachment to the Ligand unit.

In other embodiments, Linker-Drug compounds are provided for conjugationto a Ligand unit. In one embodiment, the Linker-Drug compounds aredesigned for connection to a Cell Binding Agent.

In one embodiment, the Drug Linker compound has the formula:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, G¹ is a Stretcher group (A¹) to form a connection to a        Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a        covalent bond or together with —OC(═O)— forms a self-immolative        group(s).

In another embodiment, the Drug Linker compound has the formula:G¹-L¹-L²-*

-   -   where the asterisk indicates the point of attachment to the Drug        unit, G¹ is a Stretcher unit (A¹) to form a connection to a        Ligand unit, L¹ is a Specificity unit, L² (a Spacer unit) is a        covalent bond or a self-immolative group(s).

L¹ and L² are as defined above. References to connection to A¹ can beconstrued here as referring to a connection to G¹.

In one embodiment, where L¹ comprises an amino acid, the side chain ofthat amino acid may be protected. Any suitable protecting group may beused. In one embodiment, the side chain protecting groups are removablewith other protecting groups in the compound, where present. In otherembodiments, the protecting groups may be orthogonal to other protectinggroups in the molecule, where present.

Suitable protecting groups for amino acid side chains include thosegroups described in the Novabiochem Catalog 2006/2007. Protecting groupsfor use in a cathepsin labile linker are also discussed in Dubowchik etal.

In certain embodiments of the invention, the group L¹ includes a Lysamino acid residue.

The side chain of this amino acid may be protected with a Boc or Allocprotected group. A Boc protecting group is most preferred.

The functional group G¹ forms a connecting group upon reaction with aLigand unit (e.g., a cell binding agent.

In one embodiment, the functional group G¹ is or comprises an amino,carboxylic acid, hydroxy, thiol, or maleimide group for reaction with anappropriate group on the Ligand unit. In a preferred embodiment, G¹comprises a maleimide group.

In one embodiment, the group G¹ is an alkyl maleimide group. This groupis suitable for reaction with thiol groups, particularly cysteine thiolgroups, present in the cell binding agent, for example present in anantibody.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4        or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4        or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, L²        or D, and n is 0 to 6.

In one embodiment, n is 5.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4        or 8.

In one embodiment, the group G¹ is:

-   -   where the asterisk indicates the point of attachment to L¹, n is        0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and        m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4        or 8.

In each of the embodiments above, an alternative functionality may beused in place of the

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the asterisk indicates the bond to the remaining portion        of the G group.

In one embodiment, the maleimide group is replaced with a group selectedfrom:

-   -   —C(═O)OH,    -   —OH,    -   —NH₂,    -   —SH,    -   —C(═O)CH₂X, where X is Cl, Br or I,    -   —CHO,    -   —NHNH₂    -   —C≡CH, and    -   —N₃ (azide).

In one embodiment, L¹ is present, and G¹ is —NH₂, —NHMe, —COOH, —OH or—SH.

In one embodiment, where L¹ is present, G¹ is —NH₂ or —NHMe. Eithergroup may be the N-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is —NH₂, and C is an amino acidsequence —X₁—X₂—, as defined above.

In one embodiment, L¹ is present and G¹ is COOH. This group may be theC-terminal of an L¹ amino acid sequence.

In one embodiment, L¹ is present and G¹ is OH.

In one embodiment, L¹ is present and G¹ is SH.

The group G¹ may be convertable from one functional group to another. Inone embodiment, L¹ is present and G¹ is —NH₂. This group is convertableto another group G¹ comprising a maleimide group. For example, the group—NH₂ may be reacted with an acids or an activated acid (e.g.,N-succinimide forms) of those G¹ groups comprising maleimide shownabove.

The group G¹ may therefore be converted to a functional group that ismore appropriate for reaction with a Ligand unit.

As noted above, in one embodiment, L¹ is present and G¹ is —NH₂, —NHMe,—COOH, —OH or —SH. In a further embodiment, these groups are provided ina chemically protected form. The chemically protected form is thereforea precursor to the linker that is provided with a functional group.

In one embodiment, G¹ is —NH₂ in a chemically protected form. The groupmay be protected with a carbamate protecting group. The carbamateprotecting group may be selected from the group consisting of:

-   -   Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.

Preferably, where G¹ is —NH₂, it is protected with an Alloc or Fmocgroup.

In one embodiment, where G¹ is —NH₂, it is protected with an Fmoc group.

In one embodiment, the protecting group is the same as the carbamateprotecting group of the capping group.

In one embodiment, the protecting group is not the same as the carbamateprotecting group of the capping group. In this embodiment, it ispreferred that the protecting group is removable under conditions thatdo not remove the carbamate protecting group of the capping group.

The chemical protecting group may be removed to provide a functionalgroup to form a connection to a Ligand unit. Optionally, this functionalgroup may then be converted to another functional group as describedabove.

In one embodiment, the active group is an amine. This amine ispreferably the N-terminal amine of a peptide, and may be the N-terminalamine of the preferred dipeptides of the invention.

The active group may be reacted to yield the functional group that isintended to form a connection to a Ligand unit.

In other embodiments, the Linker unit is a precursor to the Linker uithaving an active group. In this embodiment, the Linker unit comprisesthe active group, which is protected by way of a protecting group. Theprotecting group may be removed to provide the Linker unit having anactive group.

Where the active group is an amine, the protecting group may be an amineprotecting group, such as those described in Green and Wuts.

The protecting group is preferably orthogonal to other protectinggroups, where present, in the Linker unit.

In one embodiment, the protecting group is orthogonal to the cappinggroup. Thus, the active group protecting group is removable whilstretaining the capping group. In other embodiments, the protecting groupand the capping group is removable under the same conditions as thoseused to remove the capping group.

In one embodiment, the Linker unit is:

-   -   where the asterisk indicates the point of attachment to the Drug        unit, and the wavy line indicates the point of attachment to the        remaining portion of the Linker unit, as applicable or the point        of attachment to G¹. Preferably, the wavy line indicates the        point of attachment to G¹.

In one embodiment, the Linker unit is:

-   -   where the asterisk and the wavy line are as defined above.

Other functional groups suitable for use in forming a connection betweenL¹ and the Cell Binding Agent are described in WO 2005/082023.

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide,peptide and a non-peptidic agent that specifically binds to a targetmolecule. In some embodiments, the Ligand unit may be a protein,polypeptide or peptide. In some embodiments, the Ligand unit may be acyclic polypeptide. These Ligand units can include antibodies or afragment of an antibody that contains at least one targetmolecule-binding site, lymphokines, hormones, growth factors, or anyother cell binding molecule or substance that can specifically bind to atarget.

The terms “specifically binds” and “specific binding” refer to thebinding of an antibody or other protein, polypeptide or peptide to apredetermined molecule (e.g., an antigen). Typically, the antibody orother molecule binds with an affinity of at least about 1×10⁷ M⁻¹, andbinds to the predetermined molecule with an affinity that is at leasttwo-fold greater than its affinity for binding to a non-specificmolecule (e.g., BSA, casein) other than the predetermined molecule or aclosely-related molecule.

Examples of Ligand units include those agents described for use in WO2007/085930, which is incorporated herein.

In some embodiments, the Ligand unit is a Cell Binding Agent that bindsto an extracellular target on a cell. Such a Cell Binding Agent can be aprotein, polypeptide, peptide or a non-peptidic agent. In someembodiments, the Cell Binding Agent may be a protein, polypeptide orpeptide. In some embodiments, the Cell Binding Agent may be a cyclicpolypeptide. The Cell Binding Agent also may be antibody or anantigen-binding fragment of an antibody. Thus, in one embodiment, thepresent invention provides an antibody-drug conjugate (ADC).

In one embodiment the antibody is a monoclonal antibody; chimericantibody; humanized antibody; fully human antibody; or a single chainantibody. One embodiment the antibody is a fragment of one of theseantibodies having biological activity. Examples of such fragmentsinclude Fab, Fab′, F(ab′)₂ and Fv fragments.

The antibody may be a diabody, a domain antibody (DAB) or a single chainantibody.

In one embodiment, the antibody is a monoclonal antibody.

Antibodies for use in the present invention include those antibodiesdescribed in WO 2005/082023 which is incorporated herein. Particularlypreferred are those antibodies for tumour-associated antigens. Examplesof those antigens known in the art include, but are not limited to,those tumour-associated antigens set out in WO 2005/082023. See, forinstance, pages 41-55.

In some embodiments, the conjugates are designed to target tumour cellsvia their cell surface antigens. The antigens may be cell surfaceantigens which are either over-expressed or expressed at abnormal timesor cell types. Preferably, the target antigen is expressed only onproliferative cells (preferably tumour cells); however this is rarelyobserved in practice. As a result, target antigens are usually selectedon the basis of differential expression between proliferative andhealthy tissue.

Antibodies have been raised to target specific tumour related antigensincluding:

-   -   Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg,        Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA        (prostate specific membrane antigen), BCMA, E-selectin, EphB2,        Melanotransferin, Muc16 and TMEFF2.

The Ligand unit is connected to the Linker unit. In one embodiment, theLigand unit is connected to A, where present, of the Linker unit.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a thioether bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through a disulfide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an amide bond.

In one embodiment, the connection between the Ligand unit and the Linkerunit is through an ester bond.

In one embodiment, the connection between the Ligand unit and the Linkeris formed between a thiol group of a cysteine residue of the Ligand unitand a maleimide group of the Linker unit.

The cysteine residues of the Ligand unit may be available for reactionwith the functional group of the Linker unit to form a connection. Inother embodiments, for example where the Ligand unit is an antibody, thethiol groups of the antibody may participate in interchain disulfidebonds. These interchain bonds may be converted to free thiol groups bye.g. treatment of the antibody with DTT prior to reaction with thefunctional group of the Linker unit.

In some embodiments, the cysteine residue is an introduced into theheavy or light chain of an antibody. Positions for cysteine insertion bysubstitution in antibody heavy or light chains include those describedin Published U.S. Application No. 2007-0092940 and International PatentPublication WO2008070593, which are incorporated herein.

Methods of Treatment

The compounds of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a compound of formula I. The term“therapeutically effective amount” is an amount sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

A compound may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Examples of treatments and therapies include,but are not limited to, chemotherapy (the administration of activeagents, including, e.g. drugs; surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a compound of formula I, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The Compounds and Conjugates can be used to treat proliferative diseaseand autoimmune disease. The term “proliferative disease” pertains to anunwanted or uncontrolled cellular proliferation of excessive or abnormalcells which is undesired, such as, neoplastic or hyperplastic growth,whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Other cancers of interest include, but are not limitedto, haematological; malignancies such as leukemias and lymphomas, suchas non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone,mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers ofB or T cell origin.

Examples of autoimmune disease include the following: rheumatoidarthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis,allergic encephalomyelitis), psoriatic arthritis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Graves' disease, glomerulonephritis, autoimmune hepatologicaldisorder, inflammatory bowel disease (e.g., Crohn's disease),anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetesmellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of Blymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g.,rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments,the autoimmunie disorder is a T cell-mediated immunological disorder.

In some embodiments, the amount of the Conjugate administered rangesfrom about 0.01 to about 10 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.01 to about 5mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.05 to about 5 mg/kg per dose. In someembodiments, the amount of the Conjugate administered ranges from about0.1 to about 5 mg/kg per dose. In some embodiments, the amount of theConjugate administered ranges from about 0.1 to about 4 mg/kg per dose.In some embodiments, the amount of the Conjugate administered rangesfrom about 0.05 to about 3 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.1 to about 3mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.1 to about 2 mg/kg per dose.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Carbinolamines

The invention includes compounds where a solvent adds across the iminebond of the PBD moiety, which is illustrated below where the solvent iswater or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD. The balance of these equilibria depend on theconditions in which the compounds are found, as well as the nature ofthe moiety itself.

These particular compounds may be isolated in solid form, for example,by lyophilisation.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R—, S—, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

General Synthetic Routes

The synthesis of PBD compounds is extensively discussed in the followingreferences, which discussions are incorporated herein by reference:

-   a) WO 00/12508 (pages 14 to 30);-   b) WO 2005/023814 (pages 3 to 10);-   c) WO 2004/043963 (pages 28 to 29); and-   d) WO 2005/085251 (pages 30 to 39).    Synthesis Route

The compounds of the present invention, where R¹⁰ and R¹¹ form anitrogen-carbon double bond between the nitrogen and carbon atoms towhich they are bound, can be synthesised from a compound of Formula 2:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), R¹², X, X′ and R″ are asdefined for compounds of formula I, Prot^(N) is a nitrogen protectinggroup for synthesis and Prot^(O) is a protected oxygen group forsynthesis or an oxo group, by deprotecting the imine bond by standardmethods.

The compound produced may be in its carbinolamine or carbinolamine etherform depending on the solvents used. For example if Prot^(N) is Allocand Prot^(O) is an oxygen protecting group for synthesis, then thedeprotection is carried using palladium to remove the N10 protectinggroup, followed by the elimination of the oxygen protecting group forsynthesis. If Prot^(N) is Troc and Prot^(O) is an oxygen protectinggroup for synthesis, then the deprotection is carried out using a Cd/Pbcouple to yield the compound of formula (I). If Prot^(N) is SEM, or ananalogous group, and Prot^(O) is an oxo group, then the oxo group can beremoved by reduction, which leads to a protected carbinolamineintermediate, which can then be treated to remove the SEM protectinggroup, followed by the elimination of water. The reduction of thecompound of Formula 2 can be accomplished by, for example, lithiumtetraborohydride, whilst a suitable means for removing the SEMprotecting group is treatment with silica gel.

Compounds of formula 2 can be synthesised from a compound of formula 3a:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of formula 2, by coupling an organometallicderivative comprising R¹², such as an organoboron derivative. Theorganoboron derivative may be a boronate or boronic acid.

Compounds of formula 2 can be synthesised from a compound of formula 3b:

where R¹², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of formula 2, by coupling an organometallicderivative comprising R², such as an organoboron derivative. Theorganoboron derivative may be a boronate or boronic acid.

Compounds of formulae 3a and 3b can be synthesised from a compound offormula 4:

where R², R⁶, R⁷, R⁹, R^(6′), R^(7′), R^(9′), X, X′ and R″ are asdefined for compounds of formula 2, by coupling about a singleequivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallicderivative, such as an organoboron derivative, comprising R² or R¹².

The couplings described above are usually carried out in the presence ofa palladium catalyst, for example Pd(PPh₃)₄, Pd(OCOCH₃)₂, PdCl₂,Pd₂(dba)₃. The coupling may be carried out under standard conditions, ormay also be carried out under microwave conditions.

The two coupling steps are usually carried out sequentially. They may becarried out with or without purification between the two steps. If nopurification is carried out, then the two steps may be carried out inthe same reaction vessel. Purification is usually required after thesecond coupling step. Purification of the compound from the undesiredby-products may be carried out by column chromatography or ion-exchangeseparation.

The synthesis of compounds of formula 4 where Prot^(O) is an oxo groupand Prot^(N) is SEM are described in detail in WO 00/12508, which isincorporated herein by reference. In particular, reference is made toscheme 7 on page 24, where the above compound is designated asintermediate P. This method of synthesis is also described in WO2004/043963.

The synthesis of compounds of formula 4 where Prot^(O) is a protectedoxygen group for synthesis are described in WO 2005/085251, whichsynthesis is herein incorporated by reference.

Compounds of formula I where R¹⁰ and R^(10′) are H and R¹¹ and R^(11′)are SO_(z)M, can be synthesised from compounds of formula I where R¹⁰and R¹¹ form a nitrogen-carbon double bond between the nitrogen andcarbon atoms to which they are bound, by the addition of the appropriatebisulphite salt or sulphinate salt, followed by an appropriatepurification step. Further methods are described in GB 2 053 894, whichis herein incorporated by reference.

Nitrogen Protecting Groups for Synthesis

Nitrogen protecting groups for synthesis are well known in the art. Inthe present invention, the protecting groups of particular interest arecarbamate nitrogen protecting groups and hemi-aminal nitrogen protectinggroups.

Carbamate nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, Inc., 1999, which is incorporated herein by reference.

Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC,Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.

Other possible groups are nitrobenzyloxycarbonyl (e.g.4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.

Those protecting groups which can be removed with palladium catalysisare not preferred, e.g. Alloc.

Hemi-aminal nitrogen protecting groups have the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference. The groups disclosed herein can be applied to compounds ofthe present invention. Such groups include, but are not limited to, SEM,MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, Cl₃CCH₂OCH₂—.Protected Oxygen Group for Synthesis

Protected oxygen group for synthesis are well known in the art. A largenumber of suitable oxygen protecting groups are described on pages 23 to200 of Greene, T. W. and Wuts, G. M., Protective Groups in OrganicSynthesis, 3^(rd) Edition, John Wiley & Sons, Inc., 1999, which isincorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers,alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates,and sulfonates.

Preferred oxygen protecting groups include acetates, TBS and THP.

Synthesis of Drug Conjugates

Conjugates can be prepared as previously described. Linkers having amaleimidyl group (A), a peptide group (L¹) and self-immolative group(L²) can be prepared as described in U.S. Pat. No. 6,214,345. Linkershaving a maleimidyl group (A) and a peptide group (L¹) can be preparedas described in WO 2009-0117531. Other linkers can be prepared accordingto the references cited herein or as known to the skilled artisan.

Linker-Drug compounds can be prepared according to methods known in theart. Linkage of amine-based X substituents (of the PDB dimer Drug unit)to active groups of the Linker units can be performed according tomethods generally described in U.S. Pat. Nos. 6,214,345 and 7,498,298;and WO 2009-0117531, or as otherwise known to the skilled artisan.

Antibodies can be conjugated to Linker-Drug compounds as described inDoronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly,antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37°C. The progress of the reaction, which reduces interchain disulfides, ismonitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) andallowed to proceed until the desired level of thiols/mAb is achieved.The reduced antibody is then cooled to 0° C. and alkylated with 1.5equivalents of maleimide drug-linker per antibody thiol. After 1 hour,the reaction is quenched by the addition of 5 equivalents of N-acetylcysteine. Quenched drug-linker is removed by gel filtration over a PD-10column. The ADC is then sterile-filtered through a 0.22 μm syringefilter. Protein concentration can be determined by spectral analysis at280 nm and 329 nm, respectively, with correction for the contribution ofdrug absorbance at 280 nm. Size exclusion chromatography can be used todetermine the extent of antibody aggregation, and RP-HPLC can be used todetermine the levels of remaining NAC-quenched drug-linker.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In some embodiments, R^(6′), R^(7′), R^(9′), R^(10′), R^(11′) and Y′ arepreferably the same as R⁶, R⁷, R⁹, R¹⁰, R¹¹ and Y respectively.

Dimer Link

Y and Y′ are preferably O.

R″ is preferably a C₃₋₇ alkylene group with no substituents. Morepreferably R″ is a C₃, C₅ or C₇ alkylene. Most preferably, R″ is a C₃ orC₅ alkylene.

R⁶ to R⁹

R⁹ is preferably H.

R⁶ is preferably selected from H, OH, OR, SH, NH₂, nitro and halo, andis more preferably H or halo, and most preferably is H.

R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, andhalo, and more preferably independently selected from H, OH and OR,where R is preferably selected from optionally substituted C₁₋₇ alkyl,C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R may be more preferably aC₁₋₄ alkyl group, which may or may not be substituted. A substituent ofinterest is a C₅₋₆ aryl group (e.g. phenyl). Particularly preferredsubstituents at the 7-positions are OMe and OCH₂Ph. Other substituentsof particular interest are dimethylamino (i.e. —NMe₂); —(OC₂H₄)_(q)OMe,where q is from 0 to 2; nitrogen-containing C₆ heterocyclyls, includingmorpholino, piperidinyl and N-methyl-piperazinyl.

These preferences apply to R^(9′), R^(6′) and R^(7′) respectively.

R²

A in R² may be phenyl group or a C₅₋₇ heteroaryl group, for examplefuranyl, thiophenyl and pyridyl. In some embodiments, A is preferablyphenyl.

X is a group selected from the list comprising: OH, SH, CO₂H, COH,N═C═O, NHNH₂, CONHNH₂,

and NHR^(N), wherein R^(N) is selected from the group comprising H andC₁ alkyl. X may preferably be: OH, SH, CO₂H, —N═C═O or NHR^(N), and maymore preferably be: OH, SH, CO₂H, —N═C═O or NH₂. Particularly preferredgroups include: OH, SH and NH₂, with NH₂ being the most preferred group.

Q²-X may be on any of the available ring atoms of the C₅₋₇ aryl group,but is preferably on a ring atom that is not adjacent the bond to theremainder of the compound, i.e. it is preferably β or γ to the bond tothe remainder of the compound. Therefore, where the C₅₋₇ aryl group (A)is phenyl, the substituent (Q²-X) is preferably in the meta- orpara-positions, and more preferably is in the para-position.

In some embodiments, Q¹ is a single bond. In these embodiments, Q² isselected from a single bond and —Z—(CH₂)_(n)—, where Z is selected froma single bond, O, S and NH and is from 1 to 3. In some of theseembodiments, Q² is a single bond. In other embodiments, Q² is—Z—(CH₂)_(n)—. In these embodiments, Z may be O or S and n may be 1 or nmay be 2. In other of these embodiments, Z may be a single bond and nmay be 1.

In other embodiments, Q¹ is —CH═CH—.

In some embodiments, R² may be -A-CH₂—X and -A-X. In these embodiments,X may be OH, SH, CO₂H, COH and NH₂. In particularly preferredembodiments, X may be NH₂.

R¹²

R¹² is selected from:

-   (a) C₁₋₅ saturated aliphatic alkyl;-   (b) C₃₋₆ saturated cycloalkyl;-   (c)

-    wherein each of R²¹, R²² and R²³ are independently selected from H,    C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl,    where the total number of carbon atoms in the R¹² group is no more    than 5;-   (d)

-    wherein one of R^(25a) and R^(25b) is H and the other is selected    from: phenyl, which phenyl is optionally substituted by a group    selected from halo methyl, methoxy; pyridyl; and thiophenyl; and-   (e)

-    where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl;    C₂₋₃ alkynyl; cyclopropyl; phenyl, which phenyl is optionally    substituted by a group selected from halo methyl, methoxy; pyridyl;    and thiophenyl.

When R¹² is C₁₋₅ saturated aliphatic alkyl, it may be methyl, ethyl,propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl orpropyl (n-pentyl or isopropyl). In some of these embodiments, it may bemethyl. In other embodiments, it may be butyl or pentyl, which may belinear or branched.

When R¹² is C₃₋₆ saturated cycloalkyl, it may be cyclopropyl,cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may becyclopropyl.

When R¹² is

each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5. In someembodiments, the total number of carbon atoms in the R¹² group is nomore than 4 or no more than 3.

In some embodiments, one of R²¹, R²² and R²³ is H, with the other twogroups being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In other embodiments, two of R²¹, R²² and R²³ are H, with the othergroup being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In some embodiments, the groups that are not H are selected from methyland ethyl. In some of these embodiments, the groups that re not H aremethyl.

In some embodiments, R²¹ is H.

In some embodiments, R²² is H.

In some embodiments, R²³ is H.

In some embodiments, R²¹ and R²² are H.

In some embodiments, R²¹ and R²³ are H.

In some embodiments, R²² and R²³ are H.

When R¹² is

one of R^(25a) and R^(25b) is H and the other is selected from: phenyl,which phenyl is optionally substituted by a group selected from halo,methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the groupwhich is not H is optionally substituted phenyl. If the phenyl optionalsubstituent is halo, it is preferably fluoro. In some embodiment, thephenyl group is unsubstituted.

When R¹² is

R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl. Ifthe phenyl optional substituent is halo, it is preferably fluoro. Insome embodiment, the phenyl group is unsubstituted.

In some embodiments, R²⁴ is selected from H, methyl, ethyl, ethenyl andethynyl. In some of these embodiments, R²⁴ is selected from H andmethyl.

M and z

It is preferred that M and M′ are monovalent pharmaceutically acceptablecations, and are more preferably Na⁺.

z is preferably 3.

Particularly preferred compounds of the present invention are of formulaIa:

where R^(12a) is selected from:

andthe amino group is at either the meta or para positions of the phenylgroup.3^(rd) Aspect

The preferences expressed above for the first aspect may apply to thecompounds of this aspect, where appropriate.

When R¹⁰ is carbamate nitrogen protecting group, it may preferably beTeoc, Fmoc and Troc, and may more preferably be Troc.

When R¹¹ is O-Prot^(O), wherein Prot^(O) is an oxygen protecting group,Prot^(O) may preferably be TBS or THP, and may more preferably be TBS.

When R¹⁰ is a hemi-aminal nitrogen protecting group, it may preferablybe MOM, BOM or SEM, and may more preferably be SEM.

The preferences for compounds of formula I apply as appropriate to D inthe sixth aspect of the invention.

EXAMPLES

General Experimental Methods

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40-60° C.

Compound 1b was synthesised as described in WO 00/012508 (compound 210),which is herein incorporated by reference.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run usinga mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B)(formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then5% B to 95% B within 3 min. The composition was held for 0.5 min at 95%B, and then returned to 5% B in 0.3 minutes. Total gradient run timeequals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero deadvolume tee piece which passes into the mass spectrometer. Wavelengthdetection range: 220 to 400 nm. Function type: diode array (535 scans).Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm

LC/MS conditions specific for compounds protected by both a Troc and aTBDMs group: Chromatographic separation of Troc and TBDMS protectedcompounds was performed on a Waters Alliance 2695 HPLC system utilizinga Onyx Monolitic reversed-phase column (3 μm particles, 50×4.6 mm) fromPhenomenex Corp. Mobile-phase A consisted of 5% acetonitrile—95% watercontaining 0.1% formic acid, and mobile phase B consisted of 95%acetonitrile—5% water containing 0.1% formic acid. After 1 min at 5% B,the proportion of B was raised to 95% B over the next 2.5 min andmaintained at 95% B for a further 1 min, before returning to 95% A in 10s and re-equilibration for a further 50 sec, giving a total run time of5.0 min. The flow rate was maintained at 3.0 mL/min.

LC/MS conditions for Example 4: The HPLC (Waters Alliance 2695) was runusing a mobile phase of water (A) (formic acid 0.1%) and acetonitrile(B) (formic acid 0.1%). Gradient: initial composition 5% B for 2.0 minrising to 50% B over 3 min. The composition was held for 1 min at 50% B,before rising to 95% B over 1 minute. The gradient composition thendropped to 5% B over 2.5 minutes and was held at this percentage for 0.5minutes. Total gradient run time equals 10 min. Flow rate 1.5 mL/min,400 μL was split via a zero dead volume tee piece which passes into themass spectrometer. Wavelength detection range: 220 to 400 nm. Functiontype: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C1850×4.60 mm

Synthesis of Key Intermediates

(a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate](2a)

Method A: A catalytic amount of DMF (2 drops) was added to a stirredsolution of the nitro-acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride(0.95 mL, 1.36 g, 10.7 mmol) in dry THF (20 mL). The reaction mixturewas allowed to stir for 16 hours at room temperature and the solvent wasremoved by evaporation in vacuo. The resulting residue was re-dissolvedin dry THF (20 mL) and the acid chloride solution was added dropwise toa stirred mixture of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylatehydrochloride (859 mg, 4.73 mmol) and TEA (6.6 mL, 4.79 g, 47.3 mmol) inTHF (10 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogenatmosphere. The reaction mixture was allowed to warm to room temperatureand stirred for a further 3 hours after which time TLC (95:5 v/vCHCl₃/MeOH) and LC/MS (2.45 min (ES+) m/z (relative intensity) 721([M+H]^(+.), 20)) revealed formation of product. Excess THF was removedby rotary evaporation and the resulting residue was dissolved in DCM (50mL). The organic layer was washed with 1N HCl (2×15 mL), saturatedNaHCO₃ (2×15 mL), H₂O (20 mL), brine (30 mL) and dried (MgSO₄).Filtration and evaporation of the solvent gave the crude product as adark coloured oil. Purification by flash chromatography (gradientelution: 100% CHCl₃ to 96:4 v/v CHCl₃/MeOH) isolated the pure amide 2aas an orange coloured glass (840 mg, 54%).

Method B: Oxalyl chloride (9.75 mL, 14.2 g, 111 mmol) was added to astirred suspension of the nitro-acid 1a (17.3 g, 37.1 mmol) and DMF (2mL) in anhydrous DCM (200 mL). Following initial effervescence thereaction suspension became a solution and the mixture was allowed tostir at room temperature for 16 hours. Conversion to the acid chloridewas confirmed by treating a sample of the reaction mixture with MeOH andthe resulting bis-methyl ester was observed by LC/MS. The majority ofsolvent was removed by evaporation in vacuo, the resulting concentratedsolution was re-dissolved in a minimum amount of dry DCM and trituratedwith diethyl ether. The resulting yellow precipitate was collected byfiltration, washed with cold diethyl ether and dried for 1 hour in avacuum oven at 40° C. The solid acid chloride was added portionwise overa period of 25 minutes to a stirred suspension of(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (15.2 g,84.0 mmol) and TEA (25.7 mL, 18.7 g, 185 mmol) in DCM (150 mL) at −40°C. (dry ice/CH₃CN). Immediately, the reaction was complete as judged byLC/MS (2.47 min (ES⁺) m/z (relative intensity) 721 ([M+H]^(+.), 100)),the mixture was diluted with DCM (150 mL) and washed with 1N HCl (300mL), saturated NaHCO₃ (300 mL), brine (300 mL), filtered (through aphase separator) and the solvent evaporated in vacuo to give the pureproduct 2a as an orange solid (21.8 g, 82%).

Analytical Data: [α]²² _(D)=−46.1° (c=0.47, CHCl₃); ¹H NMR (400 MHz,CDCl₃) (rotamers) δ 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H),4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02(d, 2H, J=11.1 Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); ¹³C NMR (100MHz, CDCl₃) (rotamers) δ 172.4, 166.7, 154.6, 148.4, 137.2, 127.0,109.7, 108.2, 69.7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR,CHCl₃) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334,1274, 1211, 1177, 1072, 1050, 1008, 871 cm⁻¹; MS (ES⁺) m/z (relativeintensity) 721 ([M+H]^(+.), 47), 388 (80); HRMS [M+H]⁺. theoreticalC₃₁H₃₆N₄O₁₆ m/z 721.2199. found (ES⁺) m/z 721.2227.

(a)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate](2b)

Preparation from 1b according to Method B gave the pure product as anorange foam (75.5 g, 82%).

Analytical Data: (ES⁺) m/z (relative intensity) 749 ([M+H]^(+.), 100).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](3a)

Method A: A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) wasadded to a solution of the nitro-ester 2a (75 g, 104 mmol) in DMF (360mL). The suspension was hydrogenated in a Parr hydrogenation apparatusover 8 hours. Progress of the reaction was monitored by LC/MS (2.12 min(ES⁺) m/z (relative intensity) 597 ([M+H]^(+.), 100), (ES−) m/z(relative intensity) 595 ([M+H]^(+.), 100) after the hydrogen uptake hadstopped. Solid Pd/C was removed by filtration and the filtrate wasconcentrated by rotary evaporation under vacuum (below 10 mbar) at 40°C. to afford a dark oil containing traces of DMF and residual charcoal.The residue was digested in EtOH (500 mL) at 40° C. on a water bath(rotary evaporator bath) and the resulting suspension was filteredthrough celite and washed with ethanol (500 mL) to give a clearfiltrate. Hydrazine hydrate (10 mL, 321 mmol) was added to the solutionand the reaction mixture was heated at reflux. After 20 minutes theformation of a white precipitate was observed and reflux was allowed tocontinue for a further 30 minutes. The mixture was allowed to cool downto room temperature and the precipitate was retrieved by filtration,washed with diethyl ether (2*1 volume of precipitate) and dried in avacuum desiccator to provide 3a (50 g, 81%).

Method B: A solution of the nitro-ester 2a (6.80 g, 9.44 mmol) in MeOH(300 mL) was added to Raney™ nickel (4 large spatula ends of a ˜50%slurry in H₂O) and anti-bumping granules in a 3-neck round bottomedflask. The mixture was heated at reflux and then treated dropwise with asolution of hydrazine hydrate (5.88 mL, 6.05 g, 188 mmol) in MeOH (50mL) at which point vigorous effervescence was observed. When theaddition was complete (˜30 minutes) additional Raney™ nickel was addedcarefully until effervescence had ceased and the initial yellow colourof the reaction mixture was discharged. The mixture was heated at refluxfor a further 30 minutes at which point the reaction was deemed completeby TLC (90:10 v/v CHCl₃/MeOH) and LC/MS (2.12 min (ES⁺) m/z (relativeintensity) 597 ([M+H]^(+.), 100)). The reaction mixture was allowed tocool to around 40° C. and then excess nickel removed by filtrationthrough a sinter funnel without vacuum suction. The filtrate was reducedin volume by evaporation in vacuo at which point a colourlessprecipitate formed which was collected by filtration and dried in avacuum desiccator to provide 3a (5.40 g, 96%).

Analytical Data: [α]²⁷ _(D)=+404° (c=0.10, DMF); ¹H NMR (400 MHz,DMSO-d₆) δ 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H,J=3.98 Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62(dd, 2H, J=12.1, 3.60 Hz), 3.43 (dd, 2H, J=12.0, 4.72 Hz), 2.67-2.57 (m,2H), 2.26 (p, 2H, J=5.90 Hz), 1.99-1.89 (m, 2H); ¹³C NMR (100 MHz,DMSO-d₆) δ 169.1, 164.0, 149.9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8,54.4, 53.1, 33.5, 27.5; IR (ATR, neat) 3438, 1680, 1654, 1610, 1605,1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038,1018, 952, 870 cm⁻¹; MS (ES⁺) m/z (relative intensity) 619 ([M+Na]^(+.),10), 597 ([M+H]^(+.), 52), 445 (12), 326 (11); HRMS [M+H]⁺. theoreticalC₂₉H₃₂N₄O₁₀ m/z 597.2191. found (ES⁺) m/z 597.2205.

(b)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4′-benzodiazepin-5,11-dione](3b)

Preparation from 2b according to Method A gave the product as a whitesolid (22.1 g, 86%).

Analytical Data: MS (ES⁻) m/z (relative intensity) 623.3 ([M−H]⁻, 100);

(c)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](4a)

TBSCl (317 mg, 2.1 mmol) and imidazole (342 mg, 5.03 mmol) were added toa cloudy solution of the tetralactam 3a (250 mg, 0.42 mmol) in anhydrousDMF (6 mL). The mixture was allowed to stir under a nitrogen atmospherefor 3 hours after which time the reaction was deemed complete as judgedby LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M+H]^(+.),100)). The reaction mixture was poured onto ice (˜25 mL) and allowed towarm to room temperature with stirring. The resulting white precipitatewas collected by vacuum filtration, washed with H₂O, diethyl ether anddried in the vacuum desiccator to provide pure 4a (252 mg, 73%).

Analytical Data: [α]²³ _(D)=+234° (c=0.41, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H,J=5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H),2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H),0.08 (s, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 165.7, 151.4, 146.6,129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56.3, 55.7, 54.2, 35.2, 28.7,25.7, 18.0, −4.82 and −4.86; IR (ATR, CHCl₃) 3235, 2955, 2926, 2855,1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099,1033 cm⁻¹; MS (ES⁺) m/z (relative intensity) 825 ([M+H]^(+.), 62), 721(14), 440 (38); HRMS [M+H]⁺. theoretical C₄₁H₆₀N₄O₁₀Si₂ m/z 825.3921.found (ES⁺) m/z 825.3948.

(c)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](4b)

Preparation from 3b according to the above method gave the product as awhite solid (27.3 g, 93%).

Analytical Data: MS (ES⁺) m/z (relative intensity) 853.8 ([M+H]^(+.),100), (ES⁻) m/z (relative intensity) 851.6 ([M−H]^(−.), 100.

(d)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](5a)

A solution of n-BuLi (4.17 mL of a 1.6 M solution in hexane, 6.67 mmol)in anhydrous THF (10 mL) was added dropwise to a stirred suspension ofthe tetralactam 4a (2.20 g, 2.67 mmol) in anhydrous THF (30 mL) at −30°C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reactionmixture was allowed to stir at this temperature for 1 hour (now areddish orange colour) at which point a solution of SEMCl (1.18 mL, 1.11g, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise. The reactionmixture was allowed to slowly warm to room temperature and was stirredfor 16 hours under a nitrogen atmosphere. The reaction was deemedcomplete as judged by TLC (EtOAc) and LC/MS (4.77 min (ES+) m/z(relative intensity) 1085 ([M+H]^(+.), 100)). The THF was removed byevaporation in vacuo and the resulting residue dissolved in EtOAc (60mL), washed with H₂O (20 mL), brine (20 mL), dried (MgSO₄) filtered andevaporated in vacuo to provide the crude product. Purification by flashchromatography (80:20 v/v Hexane/EtOAc) gave the pure N10-SEM-protectedtetralactam 5a as an oil (2.37 g, 82%).

Analytical Data: [α]²³ _(D)=+163° (c=0.41, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98 Hz), 4.68 (d,2H, J=9.99 Hz), 4.57 (p, 2H, J=5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s,6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81 Hz),2.03-1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); ¹³CNMR (100 MHz, CDCl₃) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6,106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4,18.1, −1.24, −4.73; IR (ATR, CHCl₃) 2951, 1685, 1640, 1606, 1517, 1462,1433, 1360, 1247, 1127, 1065 cm⁻¹; MS (ES⁺) m/z (relative intensity)1113 ([M+Na]^(+.), 48), 1085 ([M+H]^(+.), 100), 1009 (5), 813 (6); HRMS[M+H]⁺. theoretical C₅₃H₈₈N₄O₁₂Si₄ m/z 1085.5548. found (ES⁺) m/z1085.5542.

(d) 1,1′-[[(Pentane1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5b)

Preparation from 4b according to the above method gave the product as apale orange foam (46.9 g, 100%), used without further purification.

Analytical Data: MS (ES⁺) m/z (relative intensity) 1114 ([M+H]^(+.),90), (ES⁻) m/z (relative intensity) 1158 ([M+2Na]⁻, 100).

(e)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](6a)

A solution of TBAF (5.24 mL of a 1.0 M solution in THF, 5.24 mmol) wasadded to a stirred solution of the bis-silyl ether 5a (2.58 g, 2.38mmol) in THF (40 mL) at room temperature. After stirring for 3.5 hours,analysis of the reaction mixture by TLC (95:5 v/v CHCl₃/MeOH) revealedcompletion of reaction. The reaction mixture was poured into a solutionof saturated NH₄Cl (100 mL) and extracted with EtOAc (3×30 mL). Thecombined organic layers were washed with brine (60 mL), dried (MgSO₄),filtered and evaporated in vacuo to provide the crude product.Purification by flash chromatography (gradient elution: 100% CHCl₃ to96:4 v/v CHCl₃/MeOH) gave the pure tetralactam 6a as a white foam (1.78g, 87%).

Analytical Data: [α]²³ _(D)=+202° (c=0.34, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0 Hz), 4.72 (d,2H, J=10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83 Hz), 4.20-4.16(m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH),2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77 Hz), 2.11-2.05 (m, 2H), 1.00-0.91(m, 4H), 0.00 (s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 169.5, 165.9, 151.3,147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67.2, 65.2, 56.5, 56.2,54.1, 35.2, 29.1, 18.4, −1.23; IR (ATR, CHCl₃) 2956, 1684, 1625, 1604,1518, 1464, 1434, 1361, 1238, 1058, 1021 cm⁻¹; MS (ES⁺) m/z (relativeintensity) 885 ([M+29]^(+.), 70), 857 ([M+H]^(+.), 100), 711 (8), 448(17); HRMS [M+H]⁺. theoretical C₄₁H₆₀N₄O₁₂Si₂ m/z 857.3819. found (ES⁺)m/z 857.3826.

(e)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](6b)

Preparation from 5b according to the above method gave the product as awhite foam (15.02 g).

Analytical Data: MS (ES⁺) m/z (relative intensity) 886 ([M+H]^(+.), 10),739.6 (100), (ES⁻) m/z (relative intensity) 884 ([M−H]^(−.), 40).

(f)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-[1-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione]](7a)

Method A: A 0.37 M sodium hypochlorite solution (142.5 mL, 52.71 mmol,2.4 eq) was added dropwise to a vigorously stirred mixture of the diol6a (18.8 g, 21.96 mmol, 1 eq), TEMPO (0.069 g, 0.44 mmol, 0.02 eq) and0.5 M potassium bromide solution (8.9 mL, 4.4 mmol, 0.2 eq) in DCM (115mL) at 0° C. The temperature was maintained between 0° C. and 5° C. byadjusting the rate of addition. The resultant yellow emulsion wasstirred at 0° C. to 5° C. for 1 hour. TLC (EtOAc) and LC/MS [3.53 min.(ES+) m/z (relative intensity) 875 ([M+Na]^(+.), 50), (ES−) m/z(relative intensity) 852 ([M−H]^(−.), 100)] indicated that reaction wascomplete.

The reaction mixture was filtered, the organic layer separated and theaqueous layer was backwashed with DCM (×2). The combined organicportions were washed with brine (×1), dried (MgSO₄) and evaporated togive a yellow foam. Purification by flash column chromatography(gradient elution 35/65 v/v n-hexane/EtOAC, 30/70 to 25/75 v/vn-hexane/EtOAC) afforded the bis-ketone 7a as a white foam (14.1 g,75%).

Sodium hypochlorite solution, reagent grade, available at chlorine10-13%, was used. This was assumed to be 10% (10 g NaClO in 100 g) andcalculated to be 1.34 M in NaClO. A stock solution was prepared fromthis by diluting it to 0.37 M with water. This gave a solution ofapproximately pH 14. The pH was adjusted to 9.3 to 9.4 by the additionof solid NaHCO₃. An aliquot of this stock was then used so as to give2.4 mol eq. for the reaction.

On addition of the bleach solution an initial increase in temperaturewas observed. The rate of addition was controlled, to maintain thetemperature between 0° C. to 5° C. The reaction mixture formed a thick,lemon yellow coloured, emulsion.

The oxidation was an adaptation of the procedure described in Thomas Feyet al, J. Org. Chem., 2001, 66, 8154-8159.

Method B: Solid TCCA (10.6 g, 45.6 mmol) was added portionwise to astirred solution of the alcohol 6a (18.05 g, 21.1 mmol) and TEMPO (123mg, 0.78 mmol) in anhydrous DCM (700 mL) at 0° C. (ice/acetone). Thereaction mixture was stirred at 0° C. under a nitrogen atmosphere for 15minutes after which time TLC (EtOAc) and LC/MS [3.57 min (ES+) m/z(relative intensity) 875 ([M+Na]^(+.), 50)] revealed completion ofreaction. The reaction mixture was filtered through celite and thefiltrate was washed with saturated aqueous NaHCO₃ (400 mL), brine (400mL), dried (MgSO₄), filtered and evaporated in vacuo to provide thecrude product. Purification by flash column chromatography (80:20 v/vEtOAc/Hexane) afforded the bis-ketone 7a as a foam (11.7 g, 65%).

Method C: A solution of anhydrous DMSO (0.72 mL, 0.84 g, 10.5 mmol) indry DCM (18 mL) was added dropwise over a period of 25 min to a stirredsolution of oxalyl chloride (2.63 mL of a 2.0 M solution in DCM, 5.26mmol) under a nitrogen atmosphere at −60° C. (liq N₂/CHCl₃). Afterstirring at −55° C. for 20 minutes, a slurry of the substrate 6a (1.5 g,1.75 mmol) in dry DCM (36 mL) was added dropwise over a period of 30 minto the reaction mixture. After stirring for a further 50 minutes at −55°C., a solution of TEA (3.42 mL, 2.49 g; 24.6 mmol) in dry DCM (18 mL)was added dropwise over a period of 20 min to the reaction mixture. Thestirred reaction mixture was allowed to warm to room temperature (˜1.5h) and then diluted with DCM (50 mL). The organic solution was washedwith 1 N HCl (2×25 mL), H₂O (30 mL), brine (30 mL) and dried (MgSO₄).Filtration and evaporation of the solvent in vacuo afforded the crudeproduct which was purified by flash column chromatography (80:20 v/vEtOAc/Hexane) to afford bis-ketone 7a as a foam (835 mg, 56%)

Analytical Data: [α]²⁰ _(D)=+291° (c=0.26, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d,2H, J=10.1 Hz), 4.60 (dd, 2H, J=9.85, 3.07 Hz), 4.31-4.18 (m, 6H),3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85 Hz),2.76 (dd, 2H, J=19.2, 9.90 Hz), 2.42 (p, 2H, J=5.77 Hz), 0.98-0.91 (m,4H), 0.00 (s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 206.8, 168.8, 165.9,151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9,52.4, 37.4, 29.0, 18.4, −1.24; IR (ATR, CHCl₃) 2957, 1763, 1685, 1644,1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm⁻¹; MS(ES+) m/z (relative intensity) 881 ([M+29]^(+.), 38), 853 ([M+H]^(+.),100), 707 (8), 542 (12); HRMS [M+H]⁺. theoretical C₄₁H₅₆N₄O₁₂Si₂ m/z853.3506. found (ES⁺) m/z 853.3502.

(f)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]](7b)

Preparation from 6b according to Method C gave the product as a whitefoam (10.5 g, 76%).

Analytical Data: MS (ES⁺) m/z (relative intensity) 882 ([M+H]^(+.), 30),735 (100), (ES⁻) m/z (relative intensity) 925 ([M+45]^(−.), 100), 880([M−H]^(−.), 70).

(g)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8a)

Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in oneportion to a vigorously stirred solution of bis-ketone 7a (6.08 g, 7.1mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile cooling bath)under a nitrogen atmosphere. Anhydrous triflic anhydride, taken from afreshly opened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injectedrapidly dropwise, while maintaining the temperature at −40° C. or below.The reaction mixture was allowed to stir at −45° C. for 1 hour at whichpoint TLC (50/50 v/v n-hexane/EtOAc) revealed the complete consumptionof starting material. The cold reaction mixture was immediately dilutedwith DCM (200 mL) and, with vigorous shaking, washed with water (1×100mL), 5% citric acid solution (1×200 mL) saturated NaHCO₃ (200 mL), brine(100 mL) and dried (MgSO₄). Filtration and evaporation of the solvent invacuo afforded the crude product which was purified by flash columnchromatography (gradient elution: 90:10 v/v n-hexane/EtOAc to 70:30 v/vn-hexane/EtOAc) to afford bis-enol triflate 8a as a yellow foam (5.5 g,70%).

Analytical Data: [α]²⁴ _(D)=+271° (c=0.18, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97 Hz), 5.51 (d,2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.69 Hz),4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H,J=16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J=5.85 Hz), 1.23-0.92 (m, 4H), 0.02(s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 162.7, 151.9, 148.0, 138.4,133.6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56.3, 30.8,29.0, 18.4, −1.25; IR (ATR, CHCl₃) 2958, 1690, 1646, 1605, 1517, 1456,1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm⁻¹; MS (ES⁺)m/z (relative intensity) 1144 ([M+28]^(+.), 100), 1117 ([M+H]^(+.), 48),1041 (40), 578 (8); HRMS [M+H]⁺. theoretical C₄₃H₅₄N₄O₁₆Si₂S₂F₆ m/z1117.2491. found (ES⁺) m/z 1117.2465.

(g)1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](8b)

Preparation from 7b according to the above method gave the bis-enoltriflate as a pale yellow foam (6.14 g, 82%).

Analytical Data: (ES+) m/z (relative intensity) 1146 ([M+H]^(+.), 85).

Example 1

(a)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(9)

Solid Pd(PPh₃)₄ (20.18 mg, 17.46 μmol) was added to a stirred solutionof the triflate 8a (975 mg, 0.87 mmol),4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)aniline (172 mg, 0.79mmol) and Na₂CO₃ (138 mg, 1.30 mmol) in toluene (13 mL) EtOH (6.5 mL)and H₂O (6.5 mL). The dark solution was allowed to stir under a nitrogenatmosphere for 24 hours, after which time analysis by TLC (EtOAc) andLC/MS revealed the formation of the desired mono-coupled product and aswell as the presence of unreacted starting material. The solvent wasremoved by rotary evaporation under reduced pressure and the resultingresidue partitioned between H₂O (100 mL) and EtOAc (100 mL), aftereventual separation of the layers the aqueous phase was extracted againwith EtOAc (2×25 mL). The combined organic layers were washed with H₂O(50 mL), brine (60 mL), dried (MgSO₄), filtered and evaporated in vacuoto provide the crude Suzuki product. The crude Suzuki product wassubjected to flash chromatography (40% EtOAc/60% Hexane→70% EtOAc, 30%Hexane). Removal of the excess eluent by rotary evaporation underreduced pressure afforded the desired product 9 (399 mg) in 43% yield.

¹H-NMR: (CDCl₃, 400 MHz) δ 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H),7.24 (d, 2H, J=8.5 Hz), 7.15 (t, 1H, J=2.0 Hz), 6.66 (d, 2H, J=8.5 Hz),5.52 (d, 2H, J=10.0 Hz), 4.77 (d, 1H, J=10.0 Hz), 4.76 (d, 1H, J=10.0Hz), 4.62 (dd, 1H, J=3.7, 11.0 Hz), 4.58 (dd, 1H, J=3.4, 10.6 Hz), 4.29(t, 4H, J=5.6 Hz), 4.00-3.85 (m, 8H), 3.80-3.60 (m, 4H), 3.16 (ddd, 1H,J=2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J=2.2, 10.5, 16.1 Hz), 2.43 (p,2H, J=5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H). ¹³C-NMR: (CDCl₃, 100 MHz)δ 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147.6, 139.6,134.8, 134.5, 127.9 (methine), 127.5, 125.1, 123.21, 121.5, 120.5(methine), 120.1 (methine), 116.4 (methine), 113.2 (methine), 108.7(methine), 79.8 (methylene), 79.6 (methylene), 68.7 (methylene), 68.5(methylene), 67.0 (methylene), 66.8 (methylene), 58.8 (methine), 58.0(methine), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3(methylene), 19.7 (methylene), 0.25 (methyl).

(b)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(10)

A suspension of the 4-anilino triflate [see Patent 33], (210 mg, 0.198mmol), methylboronic acid (50 mg, 0.835 mmol, 4.2 eq.), silver I oxide(139 mg, 0.600 mmol., 3 eq.), potassium phosphate tribasic (252 mg, 1.2eq w/w), triphenylarsine (36.7 mg, 0.12 mmol, 0.6 eq.) andbis(benzonitrile)dichloro-palladium II (11.5 mg, 0.030 mmol, 0.15 eq.)was heated at 75° C. in dry dioxane (8 mL) in a sealed tube under aninert atmosphere for 1.5 hrs. The reaction mixture was filtered throughcotton-wool and the filter pad rinsed with ethylacetate and the filtratewas evaporated under reduced pressure. The residue was purified bycolumn chromatography on silica gel with 80% EtOAc: 20% Hexane. Removalof excess eluent by rotary evaporation under reduced pressure gave theproduct as an off-white foam (100 mg, 0.11 mmol, 54% yield).

LC-MS RT 3.87 mins, 926 (M+H)

(c)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one(11)

Fresh LiBH₄ (44 mg, 2.0 mmol, 20 eq.) was added to a stirred solution ofthe SEM-dilactam (90 mg, 0.1 mmol) in THF (8 mL) at room temperature.The reaction mixture was allowed to stir for 0.5 hr, at which time LC-MSrevealed complete reaction. The reaction mixture was partitioned betweenwater (50 mL) and chloroform (100 mL). The organic phase was washed withbrine (50 mL), dried over magnesium sulphate and concentrated in vacuo.The resulting residue was treated with DCM (5 mL), EtOH (14 mL), H₂O (7mL) and silica gel (10 g). The viscous mixture was allowed to stir atroom temperature for 5 days. The mixture was filtered slowly through asinter funnel and the silica residue washed with 90% CHCl₃: 10% MeOH(˜250 mL) until UV activity faded completely from the eluent. Theorganic phase was washed with H₂O (50 mL), brine 60 mL), dried (MgSO₄),filtered and evaporated in vacuo to provide the crude material. Thecrude product was purified by flash chromatography (gradient from 100%CHCl₃: 0% MeOH to 96% CHCl₃: 4% MeOH) to provide the PBD dimer (5 mg 8%yield).

LC-MS RT 2.30 mins, 634 (M+H)

¹H-NMR (400 MHZ, CDCl₃) δ 7.80 (d, J=4.0 Hz, 1H), 7.73 (d, J=4.0 Hz,1H), 7.45 (s, 1H), 7.43 (s, 1H), 7.26 (bs, 1H), 7.14 (d, J=8.5 Hz, 1H),6.79 (s, 1H), 6.77 (s, 1H), 6.71-6.64 (m, 1H), 4.34-4.03 (m, 6H), 3.86(s, 3H), 3.85 (s 3H), 3.55-3.37 (m, 1H), 3.36-3.19 (m, 1H), 3.17-3.00(m, 1H), 2.96-2.80 (m, 1H), 1.75 (s 3H).

Example 2

(a) (11S,11aS)-2,2,2-trichloroethyl2-(3-aminophenyl)-1′-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-1′-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-2-(trifluoromethylsulphonyloxy)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepindiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate13

Solid 3-aminobenzeneboronic acid (60.3 mg) was added to a solution ofthe Troc protected bis triflate 12(Compound 44, WO 2006/111759) (600 mg,0.41 mmol), sodium carbonate (65 mg, 0.61 mmoml) and palladium tetrakistriphenylphosphine (0.012 mmol) in toluene (10.8 mL), ethanol (5.4 mL)and water (5.4 mL). The reaction mixture was allowed to stir at roomtemperature overnight. The reaction mixture was then partitioned betweenethylacetate and water. The organic layer was washed with water andbrine and dried over magnesium sulphate. Excess solvent was removed byrotary evaporation under reduced pressure and the resulting residue wassubjected to flash column chromatography (silica gel; gradient elutionEtOAc/hexane 20/80→30/70→40/60→60/40) to remove unreacted bis-triflate.Removal of excess eluent from selected fractions to afford the desiredcompound in 41% yield (230 mg, 0.163 mmol)

LC-MS RT 4.28 mins, 1411 (M+H); ¹H-NMR (400 MHZ, CDCl₃) δ 7.44 (bs, 1H),7.29 (s, 1H), 7.25 (s, 1H), 7.20 (s, 1H), 7.16 (t, J=7.9 Hz, 1H),6.84-6.73 (m, 3H), 6.70 (bs, 1H), 6.62 (dd, J=7.9, 1.7 Hz, 1H),6.66-6.58 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H),4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.17-4.07 (m, 2H),4.08-3.89 (m, 10H), 3.43-3.28 (m, 2H), 2.85 (d, J=1.65 Hz, 2H),2.07-1.90 (m, 4H), 1.78-1.63 (m, 2H), 0.94 (s, 9H), 0.90 (s, 9H), 0.30(s, 6H), 0.27 (s, 6H).

(b) (11S,11aS)-2,2,2-trichloroethyl2-(3-aminophenyl)-1′-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-1′-(tert-butyldimethylsilyloxy)-2-propenyl-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepindiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate14

Solid 1-propenyl boronic acid (7.1 mg, 0.084 mmol) was added to asolution of the Troc protected triflate 13 (73 mg, 0.052 mmol), sodiumcarbonate (18 mg, 0.17 mmol) and palladium tetrakis triphenylphosphine(3 mg) in toluene (1 mL), ethanol (0.5 mL) and water (0.5 mL). Thereaction mixture was allowed to stir at room temperature overnight. Thereaction mixture was then partitioned between ethyl acetate and water.The organic layer was washed with water and brine and dried overmagnesium sulphate. Excess solvent was removed by rotary evaporationunder reduced pressure and the resulting residue was eluted through aplug of silica gel with ethylacetate. Removal of excess eluent fromselected fractions afforded the coupled product 14 (40 mg, 0.031 mmol,59%).

LC-MS RT 4.38 mins, (1301, 1305, 1307, 1308, 1310 multiple masses due tochlorine isotopes)

(c)(S)-2-(3-aminophenyl)-8-(5-((S)-2-propenyl-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepine-8-yloxy)pentyloxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 15

Cadmium/lead couple (100 mg, Q Dong et al. Tetrahedron Letters vol 36,issue 32, 5681-5682, 1995) was added to a solution of the Suzuki product14 (40 mg, 0.029 mmol) in THF (1 mL) and ammonium acetate (1N, 1 mL) andthe reaction mixture was allowed to stir for 1 hour. The reaction wasfiltered through cotton wool to remove particulates and break-up theemulsion. The reaction mixture was partitioned between chloroform andwater, the phases separated and the aqueous phase extracted withchloroform. The combined organic layers were washed with brine and driedover magnesium sulphate. Rotary evaporation under reduced pressureyielded the crude product which was subjected to column chromatography(silica gel, 1→5% MeOH/CHCl₃). Removal of excess eluent by rotaryevaporation under reduced pressure afforded the desired imine product 15(9 mg 0.013 mmol 43%)

LC-MS RT 2.80 mins, 689 (M+H)

¹H-NMR (400 MHZ, CDCl₃) δ 7.88 (d, J=3.9 Hz, 1H), 7.82 (d, J=3.9 Hz,1H), 7.52 (s, 1H), 7.49 (s, 1H), 7.45 (bs, 1H), 7.15 (t, J=7.8 Hz, 1H),6.92 (bs, 1H), 6.84-6.76 (m, 3H), 6.72 (bs, 1H), 6.60 (dd, J=7.9, 1.9Hz, 1H), 6.26 (d, J=15.3 Hz, 1H), 5.67-5.51 (m, 1H), 4.46-4.35 (m, 1H),4.34-4.24 (m, 1H), 4.20-4.00 (m, 4H), 3.94 (s, 3H), 3.93 (s 3H),3.62-3.44 (m, 1H), 3.43-3.23 (m, 2H), 3.19-3.02 (m, 1H), 2.06-1.89 (m,4H), 1.84 (d, J=6.5 Hz, 3H), 1.76-1.62 (m, 2H).

Example 3

(a)(S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione16

Solid Pd(PPh₃)₄ (20 mg, 17.8 μmol) was added to a stirred solution ofthe triflate 8a (2.5 g, 2.24 mmol), 3-aminobenzeneboronic acid (291 mg,2.12 mmol) and Na₂CO₃ (356 mg, 3.35 mmol) in toluene (20 mL), EtOH (10mL) and H₂O (10 mL). The solution was allowed to stir under a nitrogenatmosphere for 3 hours at room temperature, after which time analysis byTLC (EtOAc) and LC/MS revealed the formation of the desired mono-coupledproduct and as well as the presence of unreacted starting material. Thesolvent was removed by rotary evaporation under reduced pressure and theresulting residue partitioned between H₂O (100 mL) and EtOAc (100 mL),after eventual separation of the layers the aqueous phase was extractedagain with EtOAc (2×25 mL). The combined organic layers were washed withH₂O (50 mL), brine (60 mL), dried (MgSO₄), filtered and evaporated invacuo to provide the crude Suzuki product. The crude Suzuki product wassubjected to flash chromatography (30% EtOAc/70% Hexane→80% EtOAc, 20%Hexane). Removal of the excess eluent by rotary evaporation underreduced pressure afforded the desired product (1 g) in 42% yield.

LC-MS, 4.17 minutes, ES⁺1060.19.

(b)(S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione 17

A suspension of the 3-anilino triflate, (50 mg, 47.2 μmol),methylboronic acid (8.47 mg, 141 μmol, 3 eq.), silver(I)oxide (21.8 mg,94.3 μmol., 2 eq.), potassium phosphate tribasic (60 mg, 1.2 eq w/w),triphenylarsine (5.78 mg, 18.9 μmol, 0.4 eq.) andbis(benzonitrile)dichloro-palladium II (1.81 mg, 4.7 μmol, 0.1 eq.) washeated at 67° C. in dry dioxane (2 mL) in a sealed tube under an inertatmosphere for 3 hrs. The reaction mixture was filtered throughcotton-wool and the filter pad rinsed with ethylacetate and the filtratewas evaporated under reduced pressure. The residue was purified bycolumn chromatography on silica gel with 80% EtOAc: 20% Hexane. Removalof excess eluent by rotary evaporation under reduced pressure gave theproduct as an off-white foam (18 mg, 19.4 μmol, 41% yield). The reactionwas subsequently repeated on a larger scale to afford 250 mg of the2-methyl product.

LC-MS 3.88 mins, 925.86 (M+H)

(c)(S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one18

Fresh LiBH₄ (20.6 mg, 0.95 mmol, 3.5 eq.) was added to a stirredsolution of the SEM-dilactam (250 mg, 0.27 mmol) in THF (4 mL) at roomtemperature. The reaction mixture was allowed to stir for 1.0 hr, atwhich time LC-MS revealed complete reaction. Excess LiBH₄ was quenchedwith acetone (c. 1 mL) at 0° C. (ice bath). The reaction mixture waspartitioned between water (50 mL) and 10% methanol in chloroform (100mL). The organic phase was washed with brine (50 mL), dried overmagnesium sulphate and concentrated in vacuo.

The resulting residue was treated with 10% methanol in chloroform (c. 50mL) and silica gel (20 g). The viscous mixture was allowed to stir atroom temperature for 5 days. The mixture was filtered slowly through asinter funnel and the silica residue washed with 90% CHCl₃: 10% MeOH(˜250 mL) until UV activity faded completely from the eluent. Theorganic phase was washed with H₂O (50 mL), brine 60 mL), dried (MgSO₄),filtered and evaporated in vacuo to provide the crude material. Thecrude product was purified by flash chromatography (gradient from 100%CHCl₃: 0% MeOH to 96% CHCl₃: 4% MeOH) to provide the PBD dimer 18.

Example 4

Part (i)

(a) Alternate Synthesis of(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(9)

Tetrakis(triphenylphosphine)palladium(0) (208 mg) was added to triflate(8a)(5 g), 4-anilineboronic acid (0.93 g) and sodium carbonate (0.62 g)in a mixture of toluene (60 mL), ethanol (30 mL) and water (10 mL). Thereaction mixture was allowed to stir for 3 days at room temperature. Thereaction mixture was washed with water, brine and dried over magnesiumsulphate. After filtration excess solvent was removed by rotaryevaporation under reduced pressure. The crude coupling product waspurified by flash column chromatography (silica gel; gradient: 100%hexane to 100% ethyl acetate). Pure fractions were combined and removalof excess eluent afforded the pure product as a solid (2.2 g, 93% yield,LC/MS 8.05 mins, m/z ES⁺1060).

(b)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(phenyl-vinyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(20)

A mixture of triflate 9 (0.5 g), trans-2-phenylvinylboronic acid (0.091g), triethylamine (0.38 g) and tetrakis(triphenylphosphine)palladium(0)(30 mL) in ethanol (3 mL), toluene (6 mL) and water (1 mL) wasirradiated with microwaves for 8 minutes at 80° C. in a sealed microwavevial. The reaction mixture was diluted with dichloromethane washed withwater and dried over magnesium sulphate. Excess solvent was removed byrotary evaporation under reduced pressure to afford the crude productwhich was used without further purification in the next reaction.Retention time 8.13 mins, ES⁺1014.13.

(c)(S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(phenyl-vinyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one(21)

A solution of superhydride in THF (1 M, 1.2 mL) was added by syringe toa solution of the crude Suzuki product (0.477 g) in THF (10 mL) at −78°C. (acetone/dry ice bath). The reaction mixture was allowed to stir at−78° C. for 20 minutes, after which time the reaction was quenched withwater. The reaction mixture was extracted with ethyl acetate and theorganic layer washed with brine and dried over magnesium sulphate.Removal of excess solvent by rotary evaporation under reduced pressureafforded the crude SEM-carbinolamine which was dissolved indichloromethane (3 mL), ethanol (6 mL) and water (1 mL) and stirred withsilica gel for 2 days. The reaction mixture was filtered excess solventevaporated by rotary evaporation under reduced pressure and the residuesubjected to flash column chromatography (3% methanol in chloroform).Pure fractions were combined and excess eluent removed by rotaryevaporation under reduced pressure to afford compound 21 (0.75 mg, 22%yield over 3 steps). Retention time 5.53 mins, ES⁺721.99.

Part (ii)

(a)(S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoicacid (23)

A suspension of dipeptide (22) (0.1 g, 0.54 mmol, 1 eq.) and6-maleimidohexanoic acid succinimide ester (0.165 g, 0.54 mmol, 1 eq.)in anhydrous DMF (5 mL) was stirred at room temperature for 24 hours atwhich time LCMS indicated 50% conversion to a new product. The reactionmixture was diluted with anhydrous DMF (5 mL) and the reaction wasallowed to continue for a further 24 hours. The solvent was evaporatedunder reduced pressure to give a colourless residue. Diethyl ether (60mL) was added and the mixture was sonicated for 5 min, the ether wasdecanted and the process was repeated (×2). The final ethereal portionwas filtered to isolate the product (23) as a white powder which wasdried under vacuum (0.105 g, 52%). Analytical Data: RT 2.28 min; MS(ES⁺) m/z (relative intensity) 382 ([M+H]^(+.), 90), MS (ES⁻) m/z(relative intensity) 380 ([M−H])^(−.), 100).

(b)6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-styryl)-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(24)

The unsymmetrical PBD dimer (21) (0.019 g, 26 μmol, 1 eq.) was added toa solution of the linker (23) (0.0121 g, 31.6 μmol, 1.2 eq.) and EEDQ(0.0098 g, 39.6 μmol, 1.5 eq.) in a mixture of anhydrous DCM/MeOH (3mL/0.5 mL) under an argon atmosphere. The resultant solution was stirredat room temperature for 5 hours at which time LCMS indicated 50%conversion to a new product. The reaction mixture was diluted withanhydrous DCM (2 mL) and the reaction was allowed to continue for afurther 18 hours. The solvent was evaporated under reduced pressure andthe residue purified by flash column chromatography [DCM 100% to DCM94%/MeOH 6% in 1% increments] to give the product as a yellow solid (5.2mg, 18%). Analytical Data: RT 3.10 min; MS (ES⁺) m/z (relativeintensity) 1085 ([M+H]^(+.), 90).

Example 5

(a)(S)-2-(4-aminophenyl)-8-(3-((S)-2-cyclopropyl-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(25)

A suspension of silver (I) oxide (0.441 g), potassium phosphate tribasic(1.187 g), triphenylarsine (0.116 g), cyclopropylboronic acid (0.206 g)and starting material 9 (0.5 g) in dioxane (15 mL) under and an argonatmosphere was heated to 71° C. A catalytic amount of palladium (II)bis(benzonitrile chloride) (0.036 g) was added and the reaction mixturewas allowed to stir for 2 hours and 10 mins at 71° C. The reactionmixture was filtered through celite and the filter pad washed with ethylacetate (400 mL). The organic solution was extracted with water (2×600mL) and brine (600 mL) and dried over magnesium sulphate. Removal oforganic solvent by rotary evaporation under reduced pressure affordedthe crude product which was purified via gravity silica gelchromatography (ethyl acetate only as eluent). Removal of excess eluentby rotary evaporation under reduced pressure afforded the product 25 asa yellow solid (145 mg, 32% yield). LCMS RT 3.92 mins, ES⁺952.06

(b)(S)-2-(4-aminophenyl)-8-(3-((S)-2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one(26)

A solution of super hydride (0.361 mL, 1M in THF) was added drop wiseover 5 minutes to a solution of the SEM dilictam 25 (0.137 g) inanhydrous tetrahydrofuran (5 mL) under an argon atmosphere at −78° C.LCMS after 35 minutes revealed that the reaction was complete and excesssuper hydride was quenched with water (4 mL) followed by brine (4 mL).The aqueous solution was extracted with a mixture ofdichloromethane/methanol (9:1, 2×16 mL) and the organic layer dried overmagnesium sulphate. Solvent was removed by rotary evaporation underreduced pressure and the crude product was taken up in a mixture ofethanol, dichloromethane and water (8:3:1, 15 mL) and treated withsilica gel. The thick suspension was allowed to stir for 4 days. Themixture was filtered through a sinter, washing withdichloromethane/methanol (9:1, 140 mL) until product ceased to beeluted. The organic layer was washed with brine (2×250 mL) and thendried over magnesium sulphate. Rotary evaporation under reduced pressureafforded the crude product which was subjected to flash columnchromatography (silica gel; gradient 100% to 5%methanol/dichloromethane). Removal of excess eluent afforded the product26 (23 mg, 25% yield). LCMS RT 2.42, ES⁺659.92

Example 6

(S)-2-(4-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-2-((trimethylsilyl)-ethynyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(27)

A mixture of 9 (0.150 g, 0.14 mmol), CuI (0.003 g, 0.014 mmol, 0.1 eq),Pd(PPh₃)₄ (0.0162 g, 0.014 mmol, 0.1 eq) and PPh₃ (0.007 g, 0.028 mmol,0.2 eq) was dissolved in piperidine (9 mL) in presence of molecularsieves under an argon atmosphere. Ethynyltrimethylsilane (0.06 ml, 0.42mmol, 3 eq) was added to the mixture at 70° C. and the reaction mixturewas allowed to stir overnight. The solvent was removed by rotaryevaporation under reduced pressure and the resulting brown solidpurified by flash column chromatography (silica gel, 90% EtOAc, 10%hexane). Compound 27 was obtained as an orange solid (0.043 g, 30%); Rf0.69 [EtOAc]; LC-MS (5 min) 4.28 min, ES⁺1008.28.

Example 7

(a)

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(30)

To a mixture of carboxylic acid 23 (8 mg, 21 umol) in 5%methanol/dichloromethane was added EEDQ (6.1 mg, 24.6 umol) and themixture was stirred for 15 minutes under nitrogen at an ambienttemperature. The resulting mixture was added to 11 (12 mg, 18.9 umol)and stirred for 3 hours under nitrogen. The reaction mixture wasaspirated directly onto a 1 mm radial chromatotron plate and eluted witha gradient of 1 to 4% methanol in dichloromethane. Product containingfractions were concentrated under reduced pressure to give 9.4 mg (50%)of 30 as a yellow solid: MS (ES⁻) m/z 997.18 (M+H)⁺.

(b)

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-(3-((S)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-amino)-3-methyl-1-oxobutan-2-yl)hexanamide(31)

Compound 31 was synthesised from compound 18 using the same method as inpart (a) with a yield of 25%.

Example 8 Determination of Free Drug In Vitro Cytotoxicity

Cells as detailed below were collected and plated in 96 well black-sidedplates at a density of 10,000 cells/well in 150 μL of medium. Serialdilutions of the test article (50 μL) were added, and incubation wascarried out for 92 hours at 37° C. After addition of test compound,cultures were incubated to 96 hours at 37° C. Resazurin (0.25 mM, 50 μL,Sigma, St. Louis, Mo.) in medium was added and incubation was continuedfor 4 h. The plates were read on a Fusion HT microplate reader (Packard,Meriden, Conn.) using an excitation wavelength of 525 nm and an emissionwavelength of 590 nm. Data from all assays were reduced using GraphPadPrism Version 4 for Windows (GraphPad Software, San Diego, Calif.). TheIC₅₀ concentrations compared to untreated control cells were determinedusing a 4 parameter curve fits.

The IC₅₀ (nM) values for compound 15 are:

L428 786-O HEL HL-60 MCF-7 IC₅₀ (nm) <0.00001 <0.00001 <0.00001 <0.000010.03

The same method was also used to determine the activity of compounds 11and 18:

IC₅₀ (nM) Caki-1 786-O TF1a MCF-7 11 0.06 0.1 0.07 0.2 18 0.6 1 0.7 2Alternative Cell Assay

Cells were plated in 150 μL growth media per well into black-sidedclear-bottom 96-well plates (Costar, Corning) and allowed to settle for1 hour in the biological cabinet before placing in the incubator at 37°C., 5% CO₂. The following day, 4× concentration of drug stocks wereprepared, and then titrated as 10-fold serial dilutions producing8-point dose curves and added at 50 μl per well in duplicate. Cells werethen incubated for 48 hours at 37° C., 5% CO₂ Cytotoxicity was measureby incubating with 100 μL Cell Titer Glo (Promega) solution for 1 hour,and then luminescence was measured on a Fusion HT plate reader (PerkinElmer). Data was processed with Excel (Microsoft) and GraphPad (Prism)to produce dose response curves and IC50 values were generated and datacollected.

IC₅₀ (nM) 786-O Caki-1 MCF-7 BxPC-3 HL-60 HEL 11 0.85 0.4 7 3 0.1 0.06

Example 9 Preparation of PDB Dimer Conjugates

Antibody-drug conjugates were prepared as previously described (seeDoronina et al., Nature Biotechnology, 21, 778-784 (2003)) or asdescribed below.

Engineered hlgG1 antibodies with introduced cysteines: CD70 antibodiescontaining a cysteine residue at position 239 of the heavy chain (h1F6d)were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA andadjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). Following a 1 hourincubation at 37° C., the reaction was cooled to 22° C. and 30equivalents of dehydroascorbic acid were added to selectively reoxidizethe native disulfides, while leaving cysteine 239 in the reduced state.The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reactionwas allowed to proceed for 1 hour at 22° C. The pH of the solution wasthen raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5equivalents of the PBD drug linker in DMSO were placed in a suitablecontainer for dilution with propylene glycol prior to addition to thereaction. To maintain solubility of the PBD drug linker, the antibodyitself was first diluted with propylene glycol to a final concentrationof 33% (e.g., if the antibody solution was in a 60 mL reaction volume,30 mL of propylene glycol was added). This same volume of propyleneglycol (30 mL in this example) was then added to the PBD drug linker asa diluent. After mixing, the solution of PBD drug linker in propyleneglycol was added to the antibody solution to effect the conjugation; thefinal concentration of propylene glycol is 50%. The reaction was allowedto proceed for 30 minutes and then quenched by addition of 5 equivalentsof N-acetyl cysteine. The ADC was then purified by ultrafiltrationthrough a 30 kD membrane. (Note that the concentration of propyleneglycol used in the reaction can be reduced for any particular PBD, asits sole purpose is to maintain solubility of the drug linker in theaqueous media.)

Example 10 Determination of Conjugate In Vitro Cytotoxicity

Cells as detailed below were collected and plated in 96 well black-sidedplates at a density of 10,000 cells/well in 150 μL of medium. Serialdilutions of the test article (50 μL) were added, and incubation wascarried out for 92 hours at 37° C. After addition of test compound,cultures were incubated to 96 hours at 37° C. Resazurin (0.25 mM, 50 μL,Sigma, St. Louis, Mo.) in medium was added and incubation was continuedfor 4 h. The plates were read on a Fusion HT microplate reader (Packard,Meriden, Conn.) using an excitation wavelength of 525 nm and an emissionwavelength of 590 nm. Data from all assays were reduced using GraphPadPrism Version 4 for Windows (GraphPad Software, San Diego, Calif.). TheIC₅₀ concentrations compared to untreated control cells were determinedusing a 4 parameter curve fits. The antibody used was a CD70 antibody(humanized 1F6; see Published U.S. Application No. 2009-148942) havingintroduced cysteine residues at amino acid heavy chain position 239(according to the EU numbering system) (indicated as h1F6d).

The IC₅₀ (nM) values for ADCs of compound 31 are:

ADCs Caki-1 786-O HL60 HEL TF1a h1F6d-31 7 36 5371 Max Inh = 50% Max Inh= (2dr/Ab) 40% Note: Maximum inhibition (Max Inhb) = % inhibition at topconcentration out of 100% untreated.

Example 11 Determination of In Vivo Cytotoxicity of Selected Conjugates

The following study was conducted in concordance with the Animal Careand Use Committee in a facility fully accredited by the Association forAssessment and Accreditation of Laboratory Animal Care. The antibodiesused were an antibody having introduced cysteine residues at position239 (S239C) in the heavy chains and conjugated to compound 31, and anonbinding control conjugated to the same compound 31.

Treatment studies were conducted in an antigen+xenograft model. Tumorcells were implanted subcutaneously into scid mice. Mice were randomizedto study groups (n=6). The ADC-compound 31 or control ADCs were dosed ipaccording to a q4dx4 schedule (as shown by the triangles on the x axis).Tumor volume as a function of time was determined using the formula(L×W2)/2. Animals were euthanized when tumor volumes reached 1000 mm³.

Referring to FIG. 1, the ADC of compound 31 was dosed at 0.1 (□), 0.3 (

) and 1 (▪) mg/kg. A nonbinding control, conjugated to compound 31, wasadministered at the same doses (0.1 (Δ), 0.3 (

) and 1(▴) mg/kg). All three doses of the Ab-compound 31 conjugate hadbetter activity than the nonbinding control conjugate. Untreated tumoursare shown by *.

The invention claimed is:
 1. A compound with the formula I:

wherein: R² is of formula III:

Where A is —C₆H₄—, X is NHR^(N), wherein R^(N) is H, and Q¹ is a singlebond, and Q² is a single bond; R¹² is selected from: (iia) methyl; (iib)cyclopropyl; (iic)

 wherein R²¹ and R²² are hydrogen and R²³ is methyl; (iid)

 wherein R^(25b) is H and R^(25a) is phenyl; and (iie)

 wherein R²⁴ is H; R⁶ and R⁹ are H; Where R is independently selectedfrom optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀aryl groups; R⁷ is OH or OR; R¹⁰ and R¹¹ form a nitrogen-carbon doublebond between the nitrogen and carbon atoms to which they are bound; R″os a C₃ alkylene group; Y and Y′ are O; R⁶, R⁷′, R⁹′ are selected fromthe same groups as R⁶, R⁷, and R⁹ respectively and R¹⁰′ and R¹¹′ are thesame as R¹⁰ and R¹¹.
 2. A compound according to claim 1, with thestructure:


3. A compound according to claim 1, wherein R¹² is methyl.
 4. A compoundaccording to claim 1, wherein R¹² is cyclopropyl.
 5. A compoundaccording to claim 1, wherein R¹² is

and R²¹ and R²² are H and R²³ is methyl.
 6. A compound according toclaim 1, wherein R¹² is

and R^(25a) is phenyl and R^(25b) is H.
 7. A compound according to claim1, wherein R¹² is

and R²⁴ is H.